CN103154033A

CN103154033A - Stable and soluble antibodies

Info

Publication number: CN103154033A
Application number: CN2011800503880A
Authority: CN
Inventors: L·博拉斯; D·尤里奇
Original assignee: Esbatech An Alcon Biomedical Research Unit LLC
Current assignee: Novartis AG
Priority date: 2010-10-22
Filing date: 2011-10-24
Publication date: 2013-06-12
Anticipated expiration: 2031-10-24
Also published as: JP2019054814A; CL2015002735A1; EP3336105B1; US20160176959A1; AU2011318179B2; BR112013010949B1; KR20130138806A; CO6741152A2; KR20190016137A; ZA201302537B; KR101635917B1; EP3336105A1; KR20170020558A; JP6245985B2; BR112013010949A2; TW201221142A; AR083495A1; RU2558298C2; UY33679A; CL2013001089A1

Abstract

The disclosure provides antibodies that are modified to reduce aggregration propensity, and methods of producing such antibodies. The present disclosure also provides particularly stable and soluble scFv antibodies and Fab fragments specific for TNF, which comprise specific light chain and heavy chain sequences that are optimized for stability, solubility, in vitro and in vivo binding of TNF, and low immunogenicity. The nucleic acids, vectors and host cells for expression of the recombinant antibodies of the disclosure, methods for isolating them and the use of said antibodies in medicine are also disclosed.

Description

Stablize and soluble antibodies

Cross

According to 35U.S.C. § 119, the application requires the U.S. Provisional Patent Application No.61/405 of submission on October 22nd, 2010, the U.S. Provisional Patent Application No.61/484 that on May 11st, 798 and 2011 submitted to, the right of priority of 749 (complete content of described U.S. Provisional Patent Application is incorporated this paper by reference into).

Invention field

The present invention relates to reduce the method for gathering tendency of antibody and modified to reduce to assemble the antibody of tendency.The invention still further relates to the antibody in conjunction with tumor necrosis factor alpha (TNF α).Particularly, the present invention relates to comprise the stable soluble antibody that reduces the modification of assembling, comprise scFv antibody and Fab fragment, it comprises specific light chain and the sequence of heavy chain that is optimized with regard to stability, solubleness and reduced immunogenicity.In addition, the present invention relates to for the method for diagnosing and/or treating the illness of TNF mediation.

Background of invention

Tumor necrosis factor alpha (TNF α is also referred to as cachectin) is the naturally occurring mammalian cytokine that is produced by many cell types (comprising monocyte and scavenger cell) responses intracellular toxin or other stimulation.TNF α is the main medium (Grell, M. wait people (1995) Cell, 83:793-802) of inflammatory, immunology and physiopathology reaction.

Soluble TNF α forms people (1988) Cell53:45-53 such as () Kriegler by cutting precursor transmembrane protein, and the 17kDa polypeptide of secretion is assembled into solubility homotrimer mixture (Smith, Deng people (1987), J.Biol.Chem.262:6951-6954; About the summary of TNFA, referring to Butler, wait people (1986), Nature320:584; Old (1986), Science230:630).This type of mixture is subsequently in conjunction with the acceptor that sees on various kinds of cell.In conjunction with producing a series of short inflammatory effectors, comprise that (i) discharges other pro-inflammatory cytokine for example interleukin-(IL)-6, IL-8 and IL-1, (ii) release matrix metalloprotease and (iii) expression of rise endothelial adhesion molecule, and also amplify inflammation and immune cascade reaction by white corpuscle being lured into extravascular tissue.

Many illnesss are relevant to the TNF alpha levels of rising, and many in them have significant medical importance.Show, TNF α is raised in many human diseases, and described disease comprises that for example rheumatoid arthritis (RA), inflammatory bowel illness comprise Crohn disease and ulcerative colitis, septicemia, congestive heart failure, bronchial asthma (asthma bronchiale) and multiple sclerosis to chronic disease.Human TNF alpha transgenic mice composing type ground produce high-caliber TNF α and produce the polyarthritis of spontaneous, destructive, similar RA (people 1991 such as Keffer, EMBO J., 10,4025-4031).Therefore TNF α is called pro-inflammatory cytokine.

The existing vital factor that is defined as clearly in the RA pathogenesis of TNF α, described RA is chronic, carrying out property and the disease that makes patient's weakness, it is characterized in that multi-joint arthritis and destruction, with heating and uncomfortable and tired general symptom.RA also causes chronic synovia inflammation, often makes progress into joint cartilage and osteoclasia.All found the TNF α of elevated levels in the patient's who suffers from RA synovia and peripheral blood.When using the TNF alpha blocker for the patient suffer from RA, they reduce inflammation, improve symptom and delay joint injury people (1999) such as (, Arthritis Rheum.42:1204-1208) McKown.

On physiology, TNF α also relevant to the protection of avoiding specific infection (people (1988) such as Cerami., Immunol.Today9:28).TNF α is discharged by the scavenger cell that is activated by the lipopolysaccharides of gram negative bacterium.Like this, TNF α is shown as development and pathogenetic vital endogenous medium (people (1989) such as Michie, the Br.J.Surg.76:670-671. that involves the endotoxin shock relevant to bacterial sepsis; The people such as Debets (1989), Second Vienna Shock Forum, p.463-466; The people such as Simpson (1989) Crit.Care Clin.5:27-47; The people such as Waage (1987) .Lancet1:355-357; The people such as Hammerle (1989) Second Vienna Shock Forum p.715-718; The people such as Debets (1989), Crit.Care Med.17:489-497; The people such as Calandra (1990), J.Infect.Dis.161:982-987; The people such as Revhaug (1988), Arch.Surg.123:162-170).

The same with other tract, also show TNF α in central nervous system, particularly played vital effect in neural inflammatory and autoimmune conditions (comprising multiple sclerosis, guillain-Barre syndrome (Guillain-Barre syndrome) and myasthenia gravis) and neural degeneration illness (comprising alzheimer's disease, Parkinson's disease and Huntington Chorea).TNF α also involves the illness of the related system of retina and muscle, comprise optic neuritis, macular degeneration, diabetic retinopathy, dermatomyositis, amyotrophic lateral sclerosis and muscular dystrophy, and involve neural damage, comprise traumatic brain injury, acute spinal cord injury and apoplexy.

Hepatitis is another kind of TNF α related inflammatory conditions, and except other triggered the factor, it also can be caused by virus infection (comprising Epstein-Barr virus, cytomegalovirus and hepatitis A-E virus).Hepatitis causes the Acute Hepatic inflammation at hepatic portal and leaflet zone, causes subsequently fibrosis and tumour progression.TNF α also can mediate the emaciation of cancer, and it causes most of cancer morbidity and mortality ratio (Tisdale M.J. (2004), Langenbecks Arch Surg.389:299-305).

The keying action that TNF α is risen in the pathology of inflammation, cellullar immunologic response and numerous disease has caused the searching to the TNF alpha-2 antagonists.A kind that is designed to treat the TNF alpha-2 antagonists of the alpha mediated disease of TNF is specific binding TNF α, thereby blocks antibody or the antibody fragment of its function.The use of Anti-tnfa antibody shows, the blocking-up reversible of TNF α is owing to the effect of TNF α, comprise the reduction of IL-1, GM-CSF, IL-6, IL-8, adhesion molecule and disorganization (people (1997) such as Feldmann, Adv.Immunol.1997:283-350).The TNF alpha specific inhibitor that has been obtained commercially recently comprises, the mono-clonal of anti-TNF alpha, gomphosis mouse-people's antibody (infliximab, Remicade ^TMCentocor Corporation/Johnson﹠amp; Johnson), it shows clinical efficacy in the treatment of RA and Crohn disease.Although obtained these progress, but still need to be used for the treatment of TNF α-associated conditions for example novel effective form or other antibody of the antibody of RA.Especially, there are urgent needs to the antibody of best-of-breed functionality character with the alpha mediated illness for the treatment of of arthritis and other TNF effectively constantly.

Summary of the invention

The invention provides antibody of comprising at least one sudden change of reduce assembling and for generation of the method for this antibody-like.

In one aspect, the invention provides the method for the gathering tendency that reduces antibody, described method comprises, introduce one or more modifications that reduce to assemble on the residue position at the variable light chain that participates in antibody and the interface between variable heavy chain, wherein displacement makes the free energy between variable light chain and variable heavy chain reduce 0.5kcal/mol at least, thereby compared to the gathering tendency of the parental generation antibody that does not have the modification that reduces to assemble, the gathering tendency of modified antibody is reduced.

In one aspect, method of the present invention comprises, introduce one or more amino-acid substitutions in the interface of the variable light chain (VL) of antibody and variable heavy chain (VH), wherein one or more displacements are on such residue position, it is selected so that the free energy between VL and VH reduces at least 10%, thereby the gathering tendency of antibody is reduced compared to parental generation antibody.Aspect concrete, the sequence of the sequence of the variable light chain of antibody and SEQ ID NO:1 has at least 65% identity.In other side, the sequence of variable heavy chain sequence and SEQ ID NO:3 or the sequence of SEQ ID NO:4 have at least 85% identity.

In some aspects, method of the present invention comprises, the residue on the residue on the AHo position 50 in the variable light chain of modified antibodies and/or AHo position 47, thus make the gathering tendency of antibody obtain to reduce compared to parental generation antibody.In other side, method of the present invention also comprises, modifies the residue on the AHo position 12,103 and 144 of variable heavy chain.

The present invention also provides the antibody with the gathering tendency that reduces, and it comprises one or more modifications that reduce to assemble.In some aspects, antibody of the present invention is Fab, Fab ', F (ab) ' 2, scFv (scFv), Fv fragment or linear antibody.In other side, the invention provides the dual specific or the bivalent molecule that comprise antibody of the present invention.

In other side, on the AHo position that is modified at variable light chain 50 that reduces to assemble.Aspect concrete, the modification that reduces to assemble comprises the arginine (R) on the AHo position 50 of variable light chain.In other side, the modification that reduces to assemble comprises, the displacement of the arginine (R) on the AHo position 50 of variable light chain to Methionin (K).

In other side, on the AHo position that is modified at variable light chain 47 that reduces to assemble.Aspect concrete, the modification that reduces to assemble comprises the arginine (R) on the AHo position 47 of variable light chain.In other side, the modification that reduces to assemble comprises, the displacement of the arginine (R) on the AHo position 47 of variable light chain to Methionin (K).

The present invention also provides the stable soluble antibody that is specific to TNF α, and it comprises specific light chain and the sequence of heavy chain that is optimized for the external and Binding in vivo of stability, solubility, TNF α and reduced immunogenicity.Described antibody is designed in order to diagnosis and/or the alpha mediated illness for the treatment of TNF.Also disclose the nucleic acid, carrier and the host cell that are used for expressing recombinant antibodies of the present invention, variable light chain and variable heavy chain, for separating of they method and described antibody in the purposes of medicine.

The present invention also provides the treatment TNF method of alpha mediated illness, comprises to the experimenter that these needs are arranged and uses the pharmaceutical composition that comprises Anti-tnfa antibody of the present invention.In some aspects, the alpha mediated illness of TNF for be selected from uveitis, Behcet's disease (Bechet's disease), the retinitis, dry eyes, glaucoma, The ocular disorders of syndrome, diabetic neuropathy, scleritis, age related macular degeneration and keratitis.

According to more detailed description and the claim of following some preferred embodiment, it is obvious that particularly preferred embodiment of the present invention will become.

Summary of drawings

Fig. 1 shows the residue position VL47 (solid line) of two kinds of different scFv molecule 34rFW1.4 (black) and 578rFW1.4 (grey) and the titration curve of VL50 (dotted line).

Fig. 2 A is presented at the concentration incubation that uses 60mg/ml under 40 ℃ and analyzes by SE-HPLC the stability of 34rFW1.4 under the condition of accelerating of measuring after 2 weeks.

Fig. 2 B is presented at the concentration incubation that uses 60mg/ml under 40 ℃ and analyzes by SE-HPLC the stability of 34rFW1.4_VLK50R_DHP under the condition of accelerating of measuring after 2 weeks.

Fig. 3 A is presented at the concentration incubation that uses 40mg/ml under 40 ℃ and analyzes by SE-HPLC the stability of 34rFW1.4 under the condition of accelerating of measuring after 2 weeks.

Fig. 3 B is presented at the concentration incubation that uses 40mg/ml under 40 ℃ and analyzes by SE-HPLC the stability of 34rFW1.4_VLK50R_DHP under the condition of accelerating of measuring after 2 weeks.

Fig. 4 A is presented at the concentration incubation that uses 20mg/ml under 40 ℃ and analyzes by SE-HPLC the stability of 34rFW1.4 under the condition of accelerating of measuring after 2 weeks.

Fig. 4 B is presented at the concentration incubation that uses 20mg/ml under 40 ℃ and analyzes by SE-HPLC the stability of 34rFW1.4_VLK50R_DHP under the condition of accelerating of measuring after 2 weeks.

Fig. 5 A is presented at the concentration incubation that uses 60mg/ml under 40 ℃ and analyzes by SE-HPLC the stability of 34rFW1.4 under the condition of accelerating of measuring after 2 weeks.

Fig. 5 B is presented at the concentration incubation that uses 60mg/ml under 40 ℃ and analyzes by SE-HPLC the stability of 34rFW1.4_VL_K50R under the condition of accelerating of measuring after 2 weeks.

Fig. 6 A is presented at the concentration incubation that uses 40mg/ml under 40 ℃ and analyzes by SE-HPLC the stability of 34rFW1.4 under the condition of accelerating of measuring after 2 weeks.

Fig. 6 B is presented at the concentration incubation that uses 40mg/ml under 40 ℃ and analyzes by SE-HPLC the stability of 34rFW1.4_VLK50R under the condition of accelerating of measuring after 2 weeks.

Fig. 7 A is presented at the concentration incubation that uses 20mg/ml under 40 ℃ and analyzes by SE-HPLC the stability of 34rFW1.4 under the condition of accelerating of measuring after 2 weeks.

Fig. 7 B is presented at the concentration incubation that uses 20mg/ml under 40 ℃ and analyzes by SE-HPLC the stability of 34rFW1.4_VLK50R under the condition of accelerating of measuring after 2 weeks.

Fig. 8 A is presented at the concentration incubation that uses 60mg/ml under 40 ℃ and analyzes by SE-HPLC the stability of 34rFW1.4 under the condition of accelerating of measuring after 2 weeks.

Fig. 8 B is presented at the concentration incubation that uses 60mg/ml under 40 ℃ and analyzes by SE-HPLC the stability of 34rFW1.4_K47R under the condition of accelerating of measuring after 2 weeks.

Fig. 9 A is presented at the concentration incubation that uses 20mg/ml under 40 ℃ and analyzes by SE-HPLC the stability of 34rFW1.4 under the condition of accelerating of measuring after 2 weeks.

Fig. 9 B is presented at the concentration incubation that uses 20mg/ml under 40 ℃ and analyzes by SE-HPLC the stability of 34rFW1.4_K47R under the condition of accelerating of measuring after 2 weeks.

Detailed Description Of The Invention

Overall purpose of the present invention is to be provided at the stable soluble antibody that has the gathering tendency that reduces in solution.In preferred embodiments, described antibody is scFv antibody or Fab fragment.Antibody of the present invention preferably comprises light chain disclosed herein and heavy chain.

The details that shows herein is only exemplary and is used for discussing for example the preferred embodiments of the invention, and such content is provided, and it is considered to the most useful and understandable description to the principle of each embodiment of the present invention and concept aspect.In this, do not plan to show structure content of the present invention in greater detail than profound understanding content essential to the invention, specification sheets makes those skilled in the art understand how to realize in practice several form of the present invention together with accompanying drawing and/or embodiment.

In order more easily to understand the present invention, as some term of giving a definition.Other definition is shown in whole detailed description.Unless by clear and change clearly, or make the meaningless or essentially no meaning of explaination (construction) when the application of connotation in the following example, define and be interpreted as standard otherwise be intended to following row in any explaination.Make it meaningless or basically in insignificant situation in the explaination of term, definition should be according to Merriam, the 3rd edition, or dictionary well known by persons skilled in the art, for example Oxford biological chemistry and molecular biology dictionary (Ed.Anthony Smith, Oxford University Press, Oxford, 2004).

As used herein, term " antibody " comprises complete antibody and any Fab (that is, " antigen-binding portion thereof ", " antigen-binding polypeptides " or " immunoconjugator ") or strand." antibody " comprises, comprises by at least two weights (H) chain of disulfide linkage interchain connection and glycoprotein or its antigen-binding portion thereof of two light (L) chains.Each heavy chain comprises variable region of heavy chain and (is abbreviated as in this article V _H) and CH.CH comprises 3 domain C H1, CH2 and CH3.Each light chain comprises variable region of light chain and (is abbreviated as in this article V _L) and constant region of light chain.Constant region of light chain comprises a domain C L.V _HAnd V _LThe district also can be subdivided into and insert the hypervariable region (being called complementary determining region (CDR)) in conservative zone (being called framework region (FR)) therebetween.Each V _HAnd V _LComprise 3 CDR and 4 FR, arrange in the following order from aminoterminal to carboxyl terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.The variable region of heavy chain and light chain comprises the binding domains with AI.But the combination of the constant region mediated immunity sphaeroprotein of antibody and host tissue or the factor (the first component (Clq) that comprises immune various cell (for example, effector cell) and classical complement system).

" antigen-binding portion thereof " of term antibody (or be called for short " antibody moiety ") refers to reservation specific binding antigen (for example, TNF) one or more fragments of ability of antibody.The antigen combined function that has shown antibody can be carried out by the fragment of full length antibody.The example that is included in the binding fragment in " antigen-binding portion thereof " of term antibody comprises, (i) Fab fragment is by V _L, V _H, the unit price fragment that forms of CL and CH1 structural domain; (ii) F (ab') ₂Fragment comprises the bivalent fragment by two Fab fragments of the disulfide bridge connects on hinge area; (iii) by V _HFd fragment with CH1 structural domain composition; (iv) by the V of the single armed of antibody _LAnd V _HThe Fv fragment that structural domain forms, (v) single domain or dAb fragment people such as (, (1989) Nature341:544-546) Ward, it is by V _HStructural domain forms; The combination of the CDR of the complementary determining region (CDR) that (vi) separates or two or more separation of (vii) optionally connecting by synthetic joint.In addition, although two structural domain V of Fv fragment _LAnd V _HBy the genes encoding that separates, but can use recombination method, connect them by synthetic joint, described joint makes them can be produced as single protein chain, wherein V _LWith V _HThe zone pairing forms monovalent molecule (being called scFv (scFv)); Referring to for example, the people such as Bird (1988) Science242:423-426; With people (1988) Proc.Natl.Acad.Sci.USA85:5879-5883 such as Huston).This type of single-chain antibody is also intended to be included in " antigen-binding portion thereof " of term antibody.This type of antibody fragment can obtain with routine techniques well known by persons skilled in the art, screens described fragment in the mode identical with complete antibody with regard to effectiveness.Antigen-binding portion thereof can utilize recombinant DNA technology or enzymatic or chemical chop by complete immunoglobulin (Ig) to produce.Antibody can have different isotypes, for example IgG (for example, IgG1, IgG2, IgG3 or IgG4 hypotype), IgA1, IgA2, IgD, IgE or IgM antibody.

Term " framework " refers to, the part of art-recognized antibody variable region, and it is present between the CDR district of a plurality of dispersions.This type of framework region is commonly referred to framework 1 to 4 (FR1, FR2, FR3 and FR4) and is provided for keeping the support of 3 CDR (so that CDR can form the antigen mating surface) in heavy chain or antibody light chain variable region in three-dimensional space.This type of framework also can be called as support, because they provided support for the presenting of CDR of a plurality of dispersions.Immunoglobulin superfamily for example other CDR of ankyrin repeat and fibronectin and framework can be used as antigen binding molecules (also referring to, for example, U.S. Patent No. 6,300,064,6,815,540 and the open case No.20040132028 of United States Patent (USP)).

Term " epi-position " or " antigenic determinant " refer to antigen (for example, the TNF) position of upper immunoglobulin (Ig) or antibodies specific combination.Epi-position comprises at least 3,4,5,6,7,8,9,10,11,12,13,14 or 15 amino acid with the space conformation of uniqueness usually.Referring to, for example, Epitope Mapping Protocols in Methods in Molecular Biology, the 66th volume, G.E.Morris, Ed. (1996).

Term " specific binding ", " selective binding ", " optionally combination " and " combination specifically " refer to the combination of antibody to the epi-position on predetermined antigens.Usually, antibody is with roughly less than 10 ^-7M is for example roughly less than 10 ^-8M, 10 ^-9M or 10 ^-10M or lower avidity (K _D) combination, as use surface plasma resonance (SPR) technical measurement in the BIACORE instrument.

Term " K _D" refer to the dissociation equilibrium constant of specific antibodies-AI.In certain embodiments, antibody more of the present invention are with less than approximately 10 ^-7M is for example less than approximately 10 ^-8M, 10 ^-9M or 10 ^-10M or even less dissociation equilibrium constant (K _D) in conjunction with TNF, for example, as use surface plasma resonance (SPR) technical measurement in the BIACORE instrument.

As used herein, " identity " refer between two peptide molecules or two nucleic acid between sequences match.When the position in two sequences of comparing is all occupied by identical base or amino acid monomer subunit (for example, if the position in each of two DNA moleculars is occupied by VITAMIN B4, or the position in each of two polypeptide is occupied by Methionin), each molecule is same on this position so." per-cent identity " between two sequences is that the number of the total matched position of two sequences multiply by 100 function again divided by the number of the position of comparing.For example, if in 10 positions in two sequences, 6 couplings are arranged, these two sequences have 60% identity so.For instance, DNA sequence dna CTGACT and CAGGTT have 50% identity (altogether in 6 positions, 3 couplings being arranged).Generally speaking, two sequence alignments are being compared when producing maximum identity.Can use the J.Mol.Biol. such as the people such as Needleman (1970) 48: the method for 443-453 (its can be easily with computer program for example Align program (DNAstar, Inc.) carry out) such comparison is provided.Per-cent identity between two aminoacid sequences also can use the E.Meyers of the ALIGN program that has been integrated into (2.0 editions) and the algorithm of W.Miller (Comput.Appl.Biosci., 4: 11-17 (1988)), utilize PAM120 weight residue table (weight residue table), 12 notch length point penalty and 4 breach point penalty to measure.In addition, Needleman and the Wunsch (J.Mol.Biol. of the GAP program (can obtain on www.gcg.com) that also can use to be integrated into the GCG software package of the per-cent identity between two aminoacid sequences 48: 444-453 (1970)) algorithm, utilize Blossum62 matrix or PAM250 matrix, and the length weight (length weight) of 16,14,12,10,8,6 or 4 breach weight (gap weight) and 1,2,3,4,5 or 6 is measured.

" similar " sequence refers to, has those sequences of identical and similar amino-acid residue when alignment, and wherein similar residue is the conservative substitution of the corresponding amino-acid residue in the canonical sequence of comparing.In this, " conservative substitution " of the residue in canonical sequence refer to, is used on physics or function and the displacement of carrying out with reference to the similar residue of residue (such as the residue with similar size, shape, electric charge, chemical property (comprising the ability that forms covalent linkage or hydrogen bond) etc.) accordingly.Therefore, " conservative substitution is modified " sequence is and canonical sequence or the different sequence that is to exist one or more conservative substitutions of wild-type sequence." per-cent similarity " between two sequences be, the number of the position that comprises coupling residue or conservative substitution that two sequences are total multiply by 100 function again divided by the number of the position of comparing.For example, have 2 to comprise conservative substitution if having in 6 couplings and 10 positions in 10 positions in two sequences, two sequences have 80% direct analogy (positive similarity) so.

As used herein, term " conserved sequence modification " is intended to refer to, negative impact or change do not comprise amino acid modified in conjunction with feature of the antibody of aminoacid sequence.This type of conserved sequence is modified and is comprised Nucleotide and amino-acid substitution, interpolation and disappearance.For example, can by standard technique known in the art for example the mutagenesis of site-directed mutagenesis and PCR mediation introduce modification.Conservative amino acid replacement comprises the displacement that wherein substitutes amino-acid residue with the amino-acid residue with similar side chain.Family with amino-acid residue of similar side chain is defined in the art.this class family comprises, (for example has basic side chain, Methionin, arginine, Histidine), acid side-chain (for example, aspartic acid, L-glutamic acid), uncharged polar side chain (for example, glycine, l-asparagine, glutamine, Serine, Threonine, tyrosine, halfcystine, tryptophane), non-polar sidechain (for example, L-Ala, α-amino-isovaleric acid, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met)), β branch side chain (for example, Threonine, α-amino-isovaleric acid, Isoleucine) and aromatic side chain (for example, tyrosine, phenylalanine, tryptophane, Histidine) amino acid.Therefore, the non-essential amino acid residue of the prediction in specific antibodies is preferably substituted by another amino-acid residue from identical side chain family.The method of identifying the Nucleotide do not eliminate the antigen combination and conservative aminoacid substitutions be known in the art (referring to, for example, the people such as Brummell, Biochem.32:1180-1187 (1993); People Protein Eng.12 (10): the 879-884 (1999) such as Kobayashi; With people Proc.Natl.Acad.Sci.USA94:412-417 (1997) such as Burks).

As used in this article, " amino acid consensus sequences " refers to, can use the matrix of the aminoacid sequence of at least two (preferably more) comparison, and allow to have breach in comparison in order to can determine the highest amino-acid residue of each position upper frequency, and the aminoacid sequence that produces.Consensus sequence be comprise on each position the most frequently occur amino acid whose that sequence.If two or more amino-acid residues appear on single position comparably, consensus sequence comprises two or all these amino acid.

Can be on different levels the aminoacid sequence of analysing protein.For example, can show conservative or mutability on single residue level, a plurality of residue level, tool a plurality of residues jaggy etc.Residue can show the conservative property of identical residue or can guard on the kind level.The example of amino acid kind comprises, polarity and uncharged R base (Serine, Threonine, l-asparagine and glutamine); Positively charged R base (Methionin, arginine and Histidine); Electronegative R base (L-glutamic acid and aspartic acid); Hydrophobicity R base (L-Ala, Isoleucine, leucine, methionine(Met), phenylalanine, tryptophane, α-amino-isovaleric acid and tyrosine); And special acid (halfcystine, glycine and proline(Pro)).Other kind it is known to the person skilled in the art that and can define with other data of structure determination or assessment substitutability.On this meaning, replaceable amino acid can refer to, can be replaced on this position and keep any amino acid of function conservative property.

Yet can there be variation in various degree in the amino acid that it should be noted that identical type on its biophysical properties.For example, it should be noted that some hydrophobicity R base (for example, L-Ala, Serine or Threonine) is than other hydrophobicity R basic (for example, α-amino-isovaleric acid or leucine) more hydrophilic (that is, having higher wetting ability or lower hydrophobicity).Relatively wetting ability or hydrophobicity can use art-recognized method (referring to, for example, the people such as Rose, Science, the people such as 229:834-838 (1985) and Cornette, J.Mol.Biol., 195:659-685 (1987)) measure.

As used herein, when with an aminoacid sequence (for example a, V _HOr V _LDuring sequence) with one or more other aminoacid sequences (for example, the one or more VH in database or VL sequence) comparison, can be with a sequence (for example a, V _HOr V _LSequence) amino acid position in is compared with " corresponding position " in one or more other aminoacid sequences.As used herein, " corresponding position " representative when with the best comparison of sequence, the equivalent site in the sequence that namely is compared during with the highest per-cent identity of acquisition or per-cent similarity when aligned sequences.

Term " nucleic acid molecule " refers to DNA molecular and RNA molecule.Nucleic acid molecule can be strand or two strands, but is preferably double-stranded DNA.When nucleic acid and another nucleotide sequence were placed in functional relationship, nucleic acid was " effectively connecting ".For example, if promotor or enhanser affect transcribing of sequence, it is connected in encoding sequence effectively.In certain embodiments, the invention provides the nucleic acid molecule of the separation of coding antibody of the present invention, variable light chain of the present invention and/or variable heavy chain of the present invention.In certain embodiments, nucleic acid molecule encoding of the present invention: comprise the polypeptide that has the variable region of light chain of at least 97% identity with SEQ ID NO:2 or SEQ ID NO:14; Comprise the polypeptide that has the variable region of heavy chain of at least 95% identity with SEQ ID NO:5; Or the antibody that has at least 96% identity with SEQ ID NO:10 or SEQ ID NO:17.

Term " carrier " refers to transport the nucleic acid molecule of connected another nucleic acid.The carrier of one type is " plasmid ", and it refers to other DNA section to be connected in circular double stranded DNA ring wherein.The carrier of another kind of type is virus vector, wherein other DNA section can be connected to enter viral genome.Some carrier can independently self-control (bacteria carrier and the additive type Mammals carrier that for example, have the bacterium replication origin) in the host cell that they are introduced.Other carrier (for example, non-add type Mammals carrier) can be integrated into the genome of host cell after introducing host cell, thereby copies with host genome.

Term " host cell " refers to introduce wherein the cell of expression vector.Host cell can comprise bacterium, microorganism, plant or zooblast.The bacterium that is easy to transform comprises the member of enterobacteriaceae (enterobacteriaceae), for example the bacterial strain of intestinal bacteria (Escherichia coli) or Salmonella (Salmonella); The member of Bacillaceae (Bacillaceae), for example subtilis (Bacillus subtilis); Streptococcus pneumoniae (Pneumococcus); Suis (Streptococcus) and Haemophilus influenzae (Haemophilus influenzae).Suitable microorganism comprises yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) and pichia spp (Pichia pastoris).Suitable animal host's clone comprises CHO (Chinese hamster ovary strain) and NS0 cell.

Term " treatment ", " treatment " and " medical treatment " refer to therapeutic or the preventive measure described herein.The method of " treatment " comprises, (for example give the experimenter for the treatment of that need to be such, suffer from the experimenter of the alpha mediated illness of TNF or finally can obtain the experimenter of such illness) use antibody of the present invention, with prevention, cure, postpone illness or recurrent illness, alleviate the severity of described illness, or improve one or more symptoms of described illness, or so that extending, experimenter's survival surpasses the time of expecting in the non-existent situation of such treatment.

Term " TNF mediation illness " typically refers to, and the morbid state relevant to TNF and/or symptom comprise outbreak, the progress of its symptom or need to continue any illness of the participation of TNF.the example of the illness of TNF mediation includes but not limited to, age related macular degeneration, neovascular glaucoma, diabetic retinopathy, retinopathy of prematurity, retinopathy of prematurity syndrome, mammary cancer, lung cancer, cancer of the stomach, the esophageal carcinoma, colorectal carcinoma, liver cancer, ovarian cancer, stupor, ovary male sex cell knurl, cervical cancer, carcinoma of endometrium, endometrial hyperplasia, endometriosis, fibrosarcoma, choriocarcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngocarcinoma, hepatoblastoma, Kaposi sarcoma (Kaposi's sarcoma), melanoma, skin carcinoma, vascular tumor, cavernous hemangioma, hemangioblastoma, carcinoma of the pancreas, retinoblastoma, astrocytoma, glioblastoma multiforme, schwannoma (Schwannoma), oligodendroglioma, medulloblastoma, neuroblastoma, rhabdosarcoma, osteogenic sarcoma, leiomyosarcoma, urinary tract cancer (urinary tract carcinomas), thyroid carcinoma, the nephroblastoma (Wilm's tumor), renal cell carcinoma, prostate cancer, the abnormal angiogenesis relevant to phakomatoss, oedema (for example, relevant to cerebral tumor), Meigs syndrome (Meigs'syndrome), rheumatoid arthritis, psoriatic and atherosclerosis.The illness of TNF mediation also comprises dry eyes and the TNF α related inflammatory patient's condition, for example eye inflammation (ocular inflammation), anaphylaxis conjunctivitis, dermatitis, rhinitis and asthma, for example, comprise those cellular change that caused by the TNF alpha active that directly or indirectly causes the TNF α related inflammatory patient's condition.In addition, the illness of TNF mediation also comprise ocular angiogenesis generation, Behcet's disease, the retinitis, glaucoma,

Syndrome, diabetic neuropathy, scleritis, keratitis and uveitis.

Term " effective dose " or " significant quantity " refer to, is enough to obtain or the amount of the effect of at least part of acquisition expectation.Term " treatment significant quantity " is defined as to be enough to cure or at least part of patient's who stops suffering from disease disease and the amount of complication thereof.To depend on the severity of illness to be treated and the immune overall status that the patient controls oneself for the effective amount of this purposes.

Term " experimenter " refers to anyone or non-human animal.For example, method and composition of the present invention can be used for treating the experimenter of the illness of suffering from the TNF mediation.

Be used for to determine the numbering system of amino acid residue position of heavy chain of antibody and variable region of light chain herein corresponding to by A.Honegger, the numbering system (AHo system) of J.Mol.Biol.309 (2001) 657-670 definition.The AHo system with by the people such as Kabat (Kabat, E.A., Deng people (1991) Sequences of Proteins of Immunological Interest, the 5th edition, U.S.Department of Health and Human Services, NIH Publication No.91-3242) conversion table between the most common system of definition is provided in A.Honegger, in J.Mol.Biol.309 (2001) 657-670.

As used herein, term " gathering " refers to cause to form the process of molecular interaction between the monomer molecule of oligomer kind/association in liquor.Can use the stability study that accelerates to assess gathering in concentrated solution under stress conditions.The stability study that accelerates is designed to by increase the speed of decomposition, gathering or the chemically modified of compound with extreme storage requirement.Usually the stability study (also referred to as coercing research) that accelerates under 40 ℃ and room temperature.This type of stability study provides the valuable information about the impact that is exposed to the envrionment conditions (also referred to as stress conditions) that exceeds normal tag storage requirement.The solution of increased protein concentration is widely used in pharmaceutical industry.The solution behavior of protein under high density can be significantly different from the solution behavior based on the diluting soln analyses and prediction because of the thermodynamics nonideality in these solution.The nonideality of observing in this type systematic is relevant to protein-protein interaction (PPI).Dissimilar power plays vital effect in the gross properties of measuring this type of PPI and degree, and its Relative Contribution is affected by solute and solvent property.The effect of PPI is driven by these intermolecular forces, to control solution characteristics, comprises physical stability and protein self-association and gathering.Concentrated solution is that wherein PPI affects those solution of the protein in solution by increasing oligomerization speed.Concentrated solution for example can have the protein concn of 10mg/ml at least.

The soluble product of this process can by analytical method for example SE-HPLC detect.As used herein, term " reduce assemble modification " refers to, compares with the parental generation antibody of describing herein, reduces the modification of the tendency that antibody assembles in liquor, for example amino-acid substitution." parental generation " antibody is to comprise and the antibody of identical sequence substantially of the corresponding antibodies with the modification that reduces to assemble.For example, parental generation antibody can have the CDR identical with the antibody of modifying, residue on AHo position 47 and/or 50 that can be in variable sequence of light chain, the identical sequence of antibody that has and modify, and different on AHo position 12,103 and 144 that also can be in the variable heavy chain sequence.Also can there be other difference, as long as parental generation antibody does not comprise the modification of the minimizing gathering that is present in the antibody of modifying according to the inventive method.

As used herein, term " interface " refers to, the interaction between two variable domains of antibody (heavy chain and light chain variable structural domain).The interface comprises the interactional amino-acid residue between direct or indirect participation variable domains.Such interaction includes but not limited to, all types of nonbondings between two structural domains interact, for example Van der Waals for, hydrogen bonding, electrostatic term (electrostatic term) and hydrophobic interaction.

Unless definition clearly in addition, whole technology used herein and scientific terminology have with the present invention under the field in the identical implication of the implication usually understood of those of ordinary skill.Although method and material similar with material to the method for describing herein or that be equal to can be used for implementing or testing the present invention, describe hereinafter appropriate means and material.In the situation that contradiction is as the criterion with this specification sheets (comprising definition).In addition, material, method and embodiment are only illustrative, and are not to be determinate.

All respects of the present invention are described in following portions in more detail.Should be understood that and arbitrarily to make up each embodiment, parameter selection and scope.In addition, depend on specific embodiment, not the definition of application choice, embodiment or scope.

In one aspect, the invention provides the α in conjunction with TNF, thereby be suitable for blocking in vivo the antibody of the function of TNF α.

In certain embodiments, antibody of the present invention is through optimizing, and it has the modification that reduces gathering with respect to parental generation antibody, thereby antibody of the present invention has the gathering tendency of minimizing compared to the antibody of parental generation/unmodified.This type of modification comprises, participates in the amino-acid substitution of variable light chain (VL) and the specific residue at variable heavy chain (VH) interface.In some embodiments, reduce the modification of assembling and comprise at least one amino-acid substitution, described amino-acid substitution reduces the free energy (compared to the free energy at the VL-VH interface of parental generation antibody) at VL-VH interface in computer (in silico) modeling method, as described in this article.This type of modification comprises the amino-acid substitution of specific residue of the free energy at contribution VL-VH interface).

In certain embodiments, the modification assembled of minimizing of the present invention comprises the displacement on AHo position 50 in the VL chain.In one embodiment, be replaced into arginine (R) on AHo position 50.In another embodiment, the arginine (R) on AHo position 50 substitutes Methionin (K).

In other embodiments, the modification assembled of minimizing of the present invention comprises the displacement on AHo position 47 in the VL chain.In one embodiment, be replaced into arginine (R) on AHo position 47.In another embodiment, the arginine (R) on AHo position 47 substitutes Methionin (K).

At Honegger, A. and

A. described the AHo numbering system in detail (2001) J MoI.Biol.309:657-670).AHo position 50 in variable light chain is corresponding to Kabat position 42.AHo position 47 in variable light chain is corresponding to Kabat position 39.At the people such as Kabat (Kabat, E.A., Deng people (1991) Sequences of Proteins of Immunological Interest, the 5th edition, U.S.Department of Health and Human Services, NIH Publication No.91-3242) described the Kabat numbering system in detail in.Conversion table between the most frequently used system of the people such as AHo system and Kabat definition is provided in A.Honegger, in J.Mol.Biol.309 (2001) 657-670.

Determine the numbering system of the amino acid residue position of heavy chain of antibody and variable region of light chain for two different being used for following conversion table is provided.At the people such as Kabat (Kabat, E.A. wait people (1991) Sequences of Proteins of Immunological Interest, the 5th edition, U.S.Department of Health and Human Services, NIH Publication No.91-3242) further described the Kabat numbering system in.At Honegger, A. and Pluckthun, A. (2001) J.Mol.Biol. 309: further described the AHo numbering system in 657-670.

The variable region of heavy chain numbering

Table 1: the conversion table of the residue position in the weight chain variable structural domain

The variable region of light chain numbering

Table 2: the conversion table of the residue position in the light chain variable structural domain

Antibody of the present invention can comprise other modification as required.For example, antibody of the present invention can comprise according to U.S. Patent application No.12/973 for example, and the method for describing in 968 reduces immunogenic amino-acid substitution in its body, and/or as the deliquescent displacement of the enhancing antibody described in WO09/155725.Therefore, in one embodiment, antibody of the present invention comprises Serine (S) on 12 (the AHo numberings) of heavy chain position; Comprising Serine (S) or Threonine (T) on 103 (the AHo numberings) of heavy chain position and/or comprising Serine (S) or Threonine (T) on 144 (the AHo numberings) of heavy chain position.In addition, antibody can comprise Serine (S) or Threonine (T) on heavy chain position 97,98 and/or 99 (AHo numbering).Preferably, antibody comprises Serine (S) on 12 (the AHo numberings) of heavy chain position, is comprising Threonine (T) on 103 (the AHo numberings) of heavy chain position and comprise Threonine (T) on 144 (the AHo numberings) of heavy chain position.

In one embodiment, antibody of the present invention comprises variable light chain:

SEQ?ID?NO:1

EIVMTQSPSTLSASVGDRVIITC(X) _n=1-50WYQQKPGRAPKLLIY(X) _n=1-50

GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC(X) _n=1-50FGQGTKLTVLG

In preferred embodiments, antibody of the present invention comprises variable light chain (CDR indicates with underscore):

SEQ?ID?NO:2:

EIVMTQSPSTLSASVGDRVIITC QSSQSVYGNIWMAWYQQKPGRAPKLLIY QASKLA

SGVPSRFSGSGSGAEFTLTISSLQPDDFATYYC QGNFNTGDRYAFGQGTKLTVLG

In another embodiment, antibody of the present invention comprises variable heavy chain:

SEQ ID NO.3: variable heavy chain framework

EVQLVESGGGLVQPGGSLRLSCTAS(X) _n=1-50WVRQAPGKGLEWVG(X) _n=1-50

RFTISRDTSKNTVYLQMNSLRAEDTAVYYCAR（X) _n=I-5OWGQGTLVTVSS

In another embodiment, antibody of the present invention comprises the variable heavy chain framework

SEQ ID NO:4: variable heavy chain framework

FVQLVESGGGLVQPGGSLRLSCTVS(X) _n=1-50WVRQAPGKGLEWVG（X) _n=1-50

RFTISKDTSKNTVYLQMNSLRAEDTAVYYCAR(X) _n=1-50WGQCTLVTVSS

In preferred embodiments, antibody of the present invention comprises variable heavy chain (CDR indicates with underscore):

SEQ?ID?NO:5:

EVQLVESGGGSVQPGGSLRLSCTAS GFTISRSYWICWVRQAPGKGLEWVG CIYGDND

ITPLYANWAKGRFTISRDTSKNTVYLQMNSLRFEDTATYYCAR LGYADYAYDLWGQG

TTVTVSS

As used herein, the X residue is the CDR insertion point.X can be any naturally occurring amino acid; Can there be at least 3 and 50 amino acid of as many as.

In one embodiment, the variable light chain framework of antibody of the present invention comprises SEQ ID NO:1 and the variable heavy chain framework comprises SEQ ID NO:3 or SEQ ID NO:4.

In another embodiment, the variable light chain framework of antibody of the present invention comprises and has at least 65% identity with SEQID NO:1, and more preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, the more preferably sequence of 99% identity.Most preferably, described sequence has arginine (R) on AHo position 50.In another embodiment, described sequence has arginine (R) on AHo position 47.

In another embodiment, the variable heavy chain framework of antibody of the present invention comprises with SEQ ID No.3 and has at least 80% identity, more preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identity, the more preferably sequence of 99% identity.Preferably, described antibody comprises Serine (S) on 12 (the AHo numberings) of heavy chain position, comprising Threonine (T) on 103 (the AHo numberings) of heavy chain position and comprise Threonine (T) on 144 (the AHo numberings) of heavy chain position.

In another embodiment, the variable light chain framework of antibody of the present invention comprises with SEQ ID NO:2 or SEQ ID NO:14 and has at least 97%, 98%, more preferably the sequence of 99% identity.

In another embodiment, the variable heavy chain framework of antibody of the present invention comprises with SEQ ID NO:5 and has at least 95%, 96%, 97%, 98%, more preferably the sequence of 99% identity.

In another embodiment, antibody of the present invention comprises with SEQ ID NO:10 or SEQ ID NO:17 and has at least 96%, 97%, 98%, more becomes to select the sequence of 99% identity.

In another embodiment, the variable light chain framework of antibody of the present invention comprises SEQ ID NO:2 or SEQ ID NO:14, and the variable heavy chain framework comprises SEQ ID NO:5.

In one embodiment, antibody of the present invention and antibody fragment are single-chain antibody (scFv) or Fab fragment.In the situation that scFv antibody can be connected in the VH structural domain with the VL structural domain with either direction by flexible joint.Suitable prior art joint is comprised of GGGGS (the SEQ ID NO:6) aminoacid sequence or its variant that repeat.In a preferred embodiment of the invention, use (GGGGS) ₄(SEQ ID NO:7) joint or derivatives thereof, but also can use 1-3 variant that repeats (people (1993) such as Holliger, Proc.Natl.Acad.Sci.USA90:6444-6448).Can be used for other joint of the present invention by the people such as Alfthan (1995), Protein Eng.8:725-731, the people such as Choi (2001), Eur.J.Immunol.31:94-106, the people such as Hu (1996), Cancer Res.56:3055-3061, the people such as Kipriyanov (1999), the people such as J.Mol.Biol.293:41-56 and Roovers (2001), Cancer Immunol.Immunother.50:51-59 is described.Arrangement can be VL-joint-VH or VH-joint-VL, and previous direction is preferred direction.In the situation that the Fab fragment, the light chain variable structural domain VL that selects is merged to the constant region of people Ig κ chain, and suitable weight chain variable structural domain VH is merged first (N-terminal) constant domain CH1 to human IgG.At C-terminal, form the interchain disulphide bridges between two constant domain.

Therefore, in one embodiment, antibody of the present invention comprises sequence:

SEQ?ID?NO:8

EIVMTQSPSTLSASVGDRVIITC(X) _n=1-5oWYQQKPGRAPKLLIY(X) _n=1-50

GVPSRFSGSGSGTEETLTISSLQPDDFATYYC(X) _n=1-50FGQGTKLTVLG

GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTAS(X) _n=1-50

WVRQAPGKGLEWVG(X) _n=1-50RFTISRDTSKNTVYLQMNS

LRAEDTAVYYCAR(X) _n=1-50WGQGTLVTVSS

In another embodiment, antibody of the present invention comprises sequence:

SEQ?ID?NO:9

EIVMTQSPSTLSASVGDRVIITC(X) _n=1-50WYQQRPGKAPKLLIY(X) _n=1-50

GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(x) _n=1-50FGQGTKLTVLG

GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVS(X) _n=1-50

WVRQAPGKGLEWVG(X) _n=1-50RFTISKDTSKNTVYLQMNSLR

AEDTAVYYCAR(X) _n=1-50WGQGTLVTVSS

In preferred embodiments, antibody of the present invention comprises sequence:

SEQ?ID?NO:10

(34rFW1.4_VL_K50R_DHP):

EIVMTQSPSTLSASVGDRVIITCQSSQSVYGNIWMAWYQQKPGRAPKLLIYQASKLA

SGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQGNFNTGDRYAFGQGTKLTVLGGG

GGSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCTASGFTISRSYWICWVR

QAPGKCLEWVGCIYGDNDITPLYANWAKGRFTISRDTSKNTVYLQMNSLRAEDTATY

YCARLGYADYAYDLWGQGTTVTVSS

The variable light chain framework of SEQ ID NO.11:FW1.4 (KI27)

EIVMTQSPSTLSASVGDRVIITC(X) _n=1-50WYQQKPGKAPKLLIY(X) _n=1-50

GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC(X) _n=1-50FGQGTKLTVLG

In another embodiment, antibody of the present invention comprises variable light chain:

The variable light chain framework of the displacement of SEQ ID NO.12:FW1.4

EIVMTQSPSTLSASVGDRVIITC(X) _n=1-50WYQQKPGKAPKLLIY(X) _n=1-50

GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(X) _n＝1-50FGQGTKLTVLG

In a further preferred embodiment, antibody of the present invention comprises sequence:

SEQ?ID?NO:13

EIVMTQSPSTLSASVGDRVIITC(X) _n=1-50WYQQRPGKAPKLLIY(X) _n=1-50

GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC(X) _n=1-50FGQGTKLTVLG

In another embodiment, antibody of the present invention comprises variable light chain (CDR indicates with underscore):

SEQ?ID?NO:14:

EIVMTQSPSTLSASVGDRVIITC QSSQSVYGNIWMAWYQQRPGKAPKLLIY QASKLA

SGVPSRFSGSGSGAEFTLTISSLQPDDFATYYC QGNFNTGDRYAFGQGTKLTVL

SEQ?ID?NO:15:

EIVMTQSPSTLSASVGDRVIITC(X) _n=1-50WYQQRPGKAPKLLIY(X) _n=1-50

GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(X) _n=1-50FGQGTKLTVLG

GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTAS(X) _n=1-50

WVRQAPGKGLEWVG(X) _n=1-50RFTISRDTSKNTVYLQMNS

LRAEDTAVYYCAR(X) _n=1-50WGQGTLVTVSS

In another embodiment, antibody of the present invention comprises sequence:

SEQ?ID?NO:16:

EIVMTQSPSTLSASVGDRVIITC(X) _n=1-50WYQQRPGKAPKLLIY(X) _n=1-50

GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(X) _n=1-50FGQGTKLTVLG

GGGGSGGCGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVS(X) _n=1-5O

WVRQAPGKCLEWVG(X) _n=1-50RFTISKDTSKNTVYLQMNSLR

AEDTAVYYCAR(X) _n=1-50WGQGTLVTVSS

In one embodiment, antibody of the present invention comprises sequence:

SEQ?ID?NO:17

(34rFW1.4_VL_K47R_DHP):

EIVMTQSPSTLSASVGDRVIITCQSSQSVYGNIWMAWYQQRPGKAPKLLIYQASKLA

SGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQGNFNTGDRYAFGQGTKLTVLGGG

GGSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCTASGFTISRSYWICWVR

QAPGKGLEWVGCIYGDNDITPLYANWAKGRFTISRDTSKNTVYLQMNSLRAEDTATY

YCARLGYADYAYDLWGQGTTVTVSS

In another embodiment, antibody of the present invention comprises sequence: SEQ ID NO:18 (34rFW1.4)

EIVMTQSPSTLSASVGDRVIITCQSSQSVYGNIWMAWYQQKPGKAPKLLIYQASKLA

SGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQGNFNTGDRYAFGQGTKLTVLGGG

GGSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCTASGFTISRSYWICWVR

OAPGKGLEWVGCIYGDNDITPLYANWAKGRFTISRDTSKNTVYLQMNSLRAEDTATY

YCARLGYADYAYDLWGQGTTVTVSS

In one embodiment, the VL of parental generation antibody is or comprises, SEQ ID NO:11 or SEQ ID NO:12 or have at least 65% identity with SEQ ID NO:11 or SEQ ID NO:12, more preferably 80%, 85%, 90%, 95%, 96%, 97%, 98%, the more preferably sequence of 99% identity.In a further preferred embodiment, the VH of parental generation antibody is or comprises, SEQ ID NO:3 or SEQ ID NO:4 or have at least 80% identity with SEQ ID NO:3 or SEQ ID NO:4, more preferably 85%, 90%, 95%, 96%, 97%, 98%, the more preferably sequence of 99% identity.

The antibody of the present invention that comprises the modification of reduce assembling preferably comprises the one or more CDR from rabbit antibody.As known in the art, rabbit CDR is different from people or rodent CDR: they can comprise cysteine residues, and described cysteine residues becomes the disulphide bridges that is connected to framework or forms S-S bridge between CDR.In addition, rabbit CDR does not belong to the canonical structure (canonical structure) of any previously known usually.

The present invention has also characterized bivalent and the bispecific molecule that comprises Anti-tnfa antibody of the present invention or its fragment.Antibody of the present invention or its antigen-binding portion thereof can be derivatized to or be connected to another kind of functional molecular, for example another kind of peptide or albumen are (for example, the part of another kind of antibody or acceptor), to produce the bispecific molecule in conjunction with at least two different binding sites or target molecule.Antibody of the present invention can be derived to or be connected to and surpass other a kind of functional molecular, the different binding sites with the generation combination over two and/or the polyspecific molecule of target molecule; This type of polyspecific molecule is also intended to be included in term used herein " bispecific molecule ".The non-limiting example of bispecific molecule comprises bispecific antibody (diabody), strand bispecific antibody and series connection antibody, and this is known to those skilled in the art.

In order to produce bispecific molecule of the present invention, antibody function of the present invention ground (for example can be connected, by chemical coupling, gene fusion, non-covalent association or alternate manner) in one or more other binding molecules, for example another kind of antibody, antibody fragment, tumour-specific or pathogen specific antigen, peptide or in conjunction with stand-in, thus bispecific molecule produced.Therefore, the present invention includes and comprise at least one and have for specific the first binding molecule of TNF α and have bispecific molecule for specific second binding molecule of one or more other target epi-positions.

In one embodiment, bispecific molecule of the present invention comprises at least one antibody or its antibody fragment (comprises for example Fab, Fab', F (ab') ₂, Fv or scFv) binding specificity.Antibody can also be light chain or heavy chain homodimer, or its any minimal segment, and for example Fv or strand construct are as what describe in the people's such as Ladner U.S. Patent No. 4,946,778 (its content is incorporated this paper by reference clearly into).

Although human monoclonal antibodies is preferred, other antibody that can be used for bispecific molecule of the present invention can be mouse, chimeric and Humanized monoclonal antibodies.

Bispecific molecule of the present invention can prepare by put together the constitutive character binding specificity with methods known in the art.For example, can produce respectively each binding specificity of bispecific molecule, subsequently it be puted together each other.When binding specificity is protein or peptide, can carries out covalency with multiple coupling agent or linking agent and put together.The example of linking agent comprises albumin A, carbodiimide, N-succinimide-S-ethanoyl-thioacetic acid (SATA), 5; 5'-dithio two (2-nitrobenzoic acids) (DTNB), ortho-, meta-or p-phenylenedimaleimide (oPDM), N-succinimide-3-(2-pyridyl dithio) propionic acid (SPDP) and 4-(N-maleimide ylmethyl) hexanaphthene-1-carboxylic acid thiosuccimide ester (sulfo-SMCC) (referring to for example, the people such as Karpovsky (1984) J.Exp.Med. 160: 1686; Liu, the people such as MA (1985) Proc.Natl.Acad.Sci.USA 82: 8648).Other method comprises Paulus (1985) Behring Ins.Mitt.No.78,118-132; The people such as Brennan (1985) Science 229: the people such as 81-83 and Glennie (1987) J.Immunol. 139: the method for describing 2367-2375).Preferably puting together agent is SATA and sulfo-SMCC, and both all can obtain from Pierce Chemical Co. (Rockford, IL).

When binding specificity is antibody, can pass through the sulfydryl bonding, for example by C-terminal hinge area or other site (no matter being naturally occurring or manually-injected) of two heavy chains, they are puted together.In particularly preferred embodiments, hinge area is modified to comprise odd number (preferred 1) sulfhydryl residue before puting together.

Perhaps, the two kinds of binding specificities of can encoding in same vehicle, and express in identical host cell and assemble.When bispecific molecule is mAb x mAb, mAb x Fab, Fab x F (ab') ₂Or during part x Fab fusion rotein, the method is particularly useful.Bispecific molecule of the present invention can be to comprise a single-chain antibody and in conjunction with the single chain molecule of determinant, or comprises two in conjunction with the strand bispecific molecule of determinant.Bispecific molecule can comprise at least two single chain molecules.In addition, bispecific molecule can be the scFv of specific binding the first target, wherein utilizes flexible joint to connect VH and the VL of described scFv, and the structural domain that provides the specific binding of the second target is provided described joint.For example in International Patent Application WO 2010/006454, suitable joint is being described.Method for the preparation of bispecific molecule is described in for example U.S. Patent number 5,260,203, U.S. Patent number 5,455,030, U.S. Patent number 4,881, and 175, U.S. Patent number 5,132,405, U.S. Patent number 5,091,513, U.S. Patent number 5,476, and 786, U.S. Patent number 5,013,653, U.S. Patent number 5,258,498 and U.S. Patent number 5,482,858 in.

Bispecific molecule can pass through for example enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), facs analysis, biological assay (for example, growth-inhibiting) or measure to verify by immunoblotting the combination of its specific target.The labelled reagent (for example, antibody) that each of this type of mensuration is specific to the target mixture by use usually detects the existence of specific objective protein-antibody complex.For example, the TNF-antibody complex can use enzyme len antibody or the antibody fragment of for example identification and specific binding antibody-TNF mixture to detect.Perhaps, mixture can detect with any of multiple other immunoassay.For example, but radiolabelled antibody, and use it for radioimmunoassay (RIA) (referring to, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, it incorporates this paper by reference into).Radio isotope can be by these class methods for example with gamma counter or scintillometer or detect with radioautography.

In yet another aspect, the invention provides method for generation of the antibody of describing herein.Provide herein for generation of the method for the antibody of the gathering tendency that has minimizing in solution based on surprising observations: interact by the antibody structure territory of regulating between light chain and heavy chain, can reduce the gathering tendency of antibody, and free energy that can be by measuring the VL/VH interface (it is defined as the difference between the energy of antibody (for example scFv) and independent variable domains) is easily predicted the sudden change of minimizing gathering.As shown in herein, can produce by the free energy that reduces between variable domains and regulate the displacement that the interactional minimizing in antibody structure territory is assembled, and not affect stability or the combination activity of antibody.

In one embodiment, method of the present invention comprises step:

(i) provide the antibody that comprises variable light chain (VL) and variable heavy chain (VH);

(ii) identify the variable light chain (VL) of one or more participation antibody and the residue position at the interface between variable heavy chain (VH); With

(iii) come modified antibodies by introduce displacement on the residue position of identifying, so that described displacement makes the free energy between VL and VH structural domain be reduced by at least 0.5kcal/mol, preferred 1.0kcal/mol at least, most preferably 2.0kcal/mol is (namely at least, have the VL of displacement and the free energy between the VH structural domain than the low 0.5kcal/mol at least of the free energy between the corresponding VL that does not comprise described amino-acid substitution and VH structural domain), thereby compare with the gathering tendency of parental generation antibody, reduce the gathering tendency of the antibody of modifying.

As outlined above, antibody can be for example Fab, Fab', F (ab) ' 2, scFv (scFv), Fv fragment, bispecific antibody, strand bispecific antibody, series connection antibody or linear antibody; In preferred embodiments, antibody is scFv (scFv).

In preferred embodiments, the evaluation (being step (ii)) of the residue position at the variable light chain of one or more participation antibody and the interface between variable heavy chain comprises, measures the free energy between the VL-VH interface.This can be by completing with known information biology program.An example of suitable information biology program is CHARMM (Chemistry at HARvard Macromolecular Mechanics).

In order to measure the free energy between the VL-VH interface, generally speaking, provide the complete atomics molecule of protein to present (molecular presentation).The free energy at interface refers in the background of implicit expression solvent method (implicit solvent method), comprises the capacity volume variance between the energy sum of the complete antibody of two variable domains VL and VH and independent structural domain.This comprises that (1) is for antibody G (a); (2) for VL G (b); (3) calculate for 3 of VH G (c) independent energy.Therefore, the free energy at interface is

G interface=G (a)-G (b)-G (c)

In one embodiment, the GBMV or the PBSA that are known in the art of implicit expression solvent method.

Free energy is measured and also can be comprised, the step of the charge distribution of simulated albumin matter.Described charge distribution can be simulated based on electrostatic force or Van der Waals for.

In order to determine suitable modification, can select the amino-acid residue at one or more participations interfaces to replace.For example, be created in the molecular model that comprises the protein of one or more displacements (for example, one or more residues being changed over L-Ala) on the position of selection, and measure the interfacial free energy that the molecule through displacement presents.If the interfacial free energy of the molecular model of displacement is lower than the interfacial free energy of initial molecular model, this amino-acid residue is selected for displacement.In the background of CHARMm, can for example utilize the sudden change of Build Mutants scheme constructs.One or more displacements in molecular model can be positioned at known participation or suspect on the position that participates in the VL/VH interface.

In one embodiment, make in the other step of the molecular model that comprises one or more displacements on selected position at the energy minimization in the zone of sudden change.Described zone can be made as 10 dusts.

In one embodiment, identified residue position for displacement is occupied by charged amino acid.

The antibody that is produced by method of the present invention can comprise any suitable variable light chain or heavy chain known in the art, preferably comprises at least one from the CDR of rabbit antibody.Some preferred variable light chain and heavy chain have been described herein.For example, antibody of the present invention can comprise: VL antibody framework, itself and SEQ ID NO:11 or SEQ ID NO:12 have at least 65% identity, more preferably 80%, 85%, 90%, 95%, 96%, 97%, 98%, more preferably 99% identity, and also comprise arginine (R) on the AHo position 47 of variable light chain and/or AHo position 50; With VH antibody framework, itself and SEQ ID NO:3 have at least 80% identity, and more preferably 85%, 90%, 95%, 96%, 97%, 98%, more preferably 99% identity.

Preferably carry out the modification of one or more residues position according to the instruction of PCT/CH2008/000285 (its integral body incorporate this paper into) by reference.In brief, for given antibody subtype, some amino acid is present on the specific residue position of antibody framework.For example,

A) for people VH3 family variable region of heavy chain, preferred amino acid is:

(i) glutamine (Q) on the amino acid position 1 of use AHo or Kabat numbering system;

(ii) glutamine (Q) on the amino acid position 6 of use AHo or Kabat numbering system;

(iii) Threonine (T) or the L-Ala (A) on the amino acid position 7 of use AHo or Kabat numbering system;

(iv) L-Ala (A), α-amino-isovaleric acid (V) or the phenylalanine (F) on the amino acid position 89 of use AHo numbering system (using the amino acid position 78 of Kabat numbering system); And/or

(v) arginine (R), glutamine (Q), Isoleucine (I), leucine (L), methionine(Met) (M) or the phenylalanine (F) on the amino acid position 103 of use AHo numbering system (using the amino acid position 89 of Kabat numbering);

B) for people VH1a family variable region of heavy chain, preferred amino acid is:

(i) L-glutamic acid (E) on the amino acid position 1 of use AHo or Kabat numbering system;

(ii) L-glutamic acid (E) on the amino acid position 6 of use AHo or Kabat numbering system;

(iii) leucine (L) on the amino acid position 12 of use AHo numbering system (using the amino acid position 11 of Kabat numbering system);

(iv) methionine(Met) (M) on the amino acid position 13 of use AHo numbering system (using the amino acid position 12 of Kabat numbering system):

(v) L-glutamic acid (E) or the glutamine (Q) on the amino acid position 14 of use AHo numbering system (using the amino acid position 13 of Kabat numbering system);

(vi) leucine (L) on the amino acid position 19 of use AHo numbering system (using the amino acid position 18 of Kabat numbering system);

(vii) Isoleucine (I) on the amino acid position 21 of use AHo numbering system (using the amino acid position 20 of Kabat numbering system);

(viii) phenylalanine (F), Serine (S), Histidine (H) or the aspartic acid (D) on the amino acid position 90 of use AHo numbering system (using the amino acid position 79 of Kabat numbering system);

(ix) aspartic acid (D) or the glutamine (Q) on the amino acid position 92 of use AHo numbering system (using the amino acid position 81 of Kabat numbering system);

(x) glycine (G), l-asparagine (N) or the Threonine (T) on the amino acid position 95 of use AHo numbering system (using the amino acid position 82b of Kabat numbering system); And/or

(xi) Threonine (T), L-Ala (A), proline(Pro) (P) or the phenylalanine (F) on the amino acid position 98 of use AHo numbering system (using the amino acid position 84 of Kabat numbering system);

C) for people VH1b family variable region of heavy chain, preferred amino acid is:

(ii) Threonine (T), proline(Pro) (P), α-amino-isovaleric acid (V) or the aspartic acid (D) on the amino acid position 10 of use AHo numbering system (using the amino acid position 9 of Kabat numbering system);

(iv) α-amino-isovaleric acid (V), arginine (R), glutamine (Q) or the methionine(Met) (M) on the amino acid position 13 of use AHo numbering system (using the amino acid position 12 of Kabat numbering system):

(v) L-glutamic acid (E), arginine (R) or the methionine(Met) (M) on the amino acid position 14 of use AHo numbering system (using the amino acid position 13 of Kabat numbering system);

(vi) arginine (R), Threonine (T) or the l-asparagine (N) on the amino acid position 20 of use AHo numbering system (using the amino acid position 19 of Kabat numbering system);

(vii) Isoleucine (I), phenylalanine (F) or the leucine (L) on the amino acid position 21 of use AHo numbering system (using the amino acid position 20 of Kabat numbering system);

(viii) Methionin (K) on the amino acid position 45 of use AHo numbering system (using the amino acid position 38 of Kabat numbering system);

(ix) Threonine (T), proline(Pro) (P), α-amino-isovaleric acid (V) or the arginine (R) on the amino acid position 47 of use AHo numbering system (using the amino acid position 40 of Kabat numbering system);

(x) Methionin (K), Histidine (H) or the L-glutamic acid (E) on the amino acid position 50 of use AHo numbering system (using the amino acid position 43 of Kabat numbering system);

(xi) Isoleucine (I) on the amino acid position 55 of use AHo numbering system (using the amino acid position 48 of Kabat numbering system);

(xii) Methionin (K) on the amino acid position 77 of use AHo numbering system (using the amino acid position 66 of Kabat numbering system);

(xiii) L-Ala (A), leucine (L) or the Isoleucine (I) on the amino acid position 78 of use AHo numbering system (using the amino acid position 67 of Kabat numbering system);

(xiv) L-glutamic acid (E), Threonine (T) or the L-Ala (A) on the amino acid position 82 of use AHo numbering system (using the amino acid position 71 of Kabat numbering system);

(xv) Threonine (T), Serine (S) or the leucine (L) on the amino acid position 86 of use AHo numbering system (using the amino acid position 75 of Kabat numbering system);

(xvi) aspartic acid (D), l-asparagine (N) or the glycine (G) on the amino acid position 87 of use AHo numbering system (using the amino acid position 76 of Kabat numbering system); And/or

(xvii) l-asparagine (N) or the Serine (S) on the amino acid position 107 of use AHo numbering system (using the amino acid position 93 of Kabat numbering system);

D) for people V κ 1 family's variable region of light chain, preferred amino acid is:

(i) L-glutamic acid (E) or the Isoleucine (I) on the amino acid position 1 of use AHo or Kabat numbering system;

(ii) α-amino-isovaleric acid (V) or the Isoleucine (I) on the amino acid position 3 of use AHo or Kabat numbering system;

(iii) α-amino-isovaleric acid (V), leucine (L) or the Isoleucine (I) on the amino acid position 4 of use AHo or Kabat numbering system;

(iv) glutamine (Q) on the amino acid position 24 of use AHo or Kabat numbering system;

(v) arginine (R) or the Isoleucine (I) on the amino acid position 47 of use AHo numbering system (using the amino acid position 39 of Kabat numbering system);

(vi) arginine (R), L-glutamic acid (E), Threonine (T), methionine(Met) (M) or the glutamine (Q) on the amino acid position 50 of use AHo numbering system (using the amino acid position 42 of Kabat numbering system);

(vii) Histidine (H), Serine (S) or the phenylalanine (F) on the amino acid position 57 of use AHo numbering system (using the amino acid position 49 of Kabat numbering system);

(viii) phenylalanine (F) on the amino acid position 91 of use AHo numbering system (using the amino acid position 73 of Kabat numbering system); And/or

(ix) α-amino-isovaleric acid (V), Serine (S), glycine (G) or the Isoleucine (I) on the amino acid position 103 of use AHo numbering system (using the amino acid position 85 of Kabat numbering system);

E) for people V κ 3 family's variable region of light chain, preferred amino acid is:

(i) Threonine (T) on the amino acid position 2 of use AHo or Kabat numbering system;

(ii) Threonine (T) on the amino acid position 3 of use AHo or Kabat numbering system;

(iii) Isoleucine (I) on the amino acid position 10 of use AHo or Kabat numbering system;

(iv) tyrosine (Y) on the amino acid position 12 of use AHo or Kabat numbering system;

(v) Serine (S) on the amino acid position 18 of use AHo or Kabat numbering system;

(vi) L-Ala (A) on the amino acid position 20 of use AHo or Kabat numbering system;

(vii) methionine(Met) (M) on the amino acid position 56 of use AHo numbering system (using the amino acid position 48 of Kabat numbering system);

(viii) α-amino-isovaleric acid (V) or the Threonine (T) on the amino acid position 74 of use AHo numbering system (using the amino acid position 58 of Kabat numbering system);

(ix) l-asparagine (N) on the amino acid position 94 of use AHo numbering system (using the amino acid position 76 of Kabat numbering system);

(x) tyrosine (Y) or the Serine (S) on the amino acid position 101 of use AHo numbering system (using the amino acid position 83 of Kabat numbering system); And/or

(xi) leucine (L) or the L-Ala (A) on the amino acid position 103 of use AHo numbering system (using the amino acid position 85 of Kabat numbering system);

F) for people V λ 1 family's variable region of light chain, preferred amino acid is:

(i) leucine (L), Serine (S) or the L-glutamic acid (E) on the amino acid position 1 of use AHo or Kabat numbering system;

(ii) L-Ala (A), proline(Pro) (P), Isoleucine (I) or the tyrosine (Y) on the amino acid position 2 of use AHo or Kabat numbering system;

(iii) α-amino-isovaleric acid (V) or the methionine(Met) (M) on the amino acid position 4 of use AHo or Kabat numbering system;

(iv) L-glutamic acid (E) on the amino acid position 7 of use AHo or Kabat numbering system;

(v) L-Ala (A) on the amino acid position 11 of use AHo or Kabat numbering system;

(vi) Threonine (T) or the Serine (S) on the amino acid position 14 of use AHo or Kabat numbering system;

(vii) Histidine (H) on the amino acid position 46 of use AHo numbering system (using the amino acid position 38 of Kabat numbering system);

(viii) Threonine (T), Serine (S), l-asparagine (N), glutamine (Q) or the proline(Pro) (P) on the amino acid position 53 of use AHo numbering system (using the amino acid position 45 of Kabat numbering system);

(ix) arginine (R) or the glutamine (Q) on the amino acid position 82 of use AHo numbering system (using the amino acid position 66 of Kabat numbering system);

(x) glycine (G), Threonine (T) or the aspartic acid (D) on the amino acid position 92 of use AHo numbering system (using the amino acid position 74 of Kabat numbering system); And/or

(xi) α-amino-isovaleric acid (V), Threonine (T), Histidine (H) or the L-glutamic acid (E) on the amino acid position 103 of use AHo numbering system (using the amino acid position 85 of Kabat numbering system).

Therefore, the displacement that produces in present method is preferably in accordance with the instruction of PCT/CH2008/000285.It is known to those skilled in the art that hypotype is measured.

In one embodiment, method of the present invention comprises, at variable light chain, and modified antibodies on the AHo position 47 and/or 50 of V κ 1 variable light chain especially.Preferably, antibody is modified to comprise arginine (R) on the AHo position 47 of variable light chain and/or AHo position 50.In some embodiments, Methionin (K) is replaced by arginine (R) on the AHo position 47 of variable light chain and/or AHo position 50.Because antibody of the present invention can comprise other modification on demand, therefore, method of the present invention can comprise that modified antibodies is (for example to comprise Serine (S) on 12 (the AHo numberings) of heavy chain position; Comprising Serine (S) or Threonine (T) on 103 (the AHo numberings) of heavy chain position and/or comprising Serine (S) or Threonine (T) on 144 (the AHo numbering) of heavy chain position) other step.In addition, antibody can be modified to comprise Serine (S) or Threonine (T) on heavy chain position 97,98 and/or 99 (AHo numbering).Preferably, described method comprises modified antibodies comprising Serine (S) on 12 (the AHo numberings) of heavy chain position, is comprising Threonine (T) on 103 (the AHo numberings) of heavy chain position and comprise the step of Threonine (T) on 144 (the AHo numberings) of heavy chain position.

The present invention also provides the method that is created in the humanized antibody that has low gathering tendency in solution, and described method comprises, is chosen in the variable light chain framework that comprises arginine (R) on AHo position 47 and/or AHo position 50.Described method also can comprise, selects the variable heavy chain framework, and described framework comprises Serine (S) on 12 (the AHo numberings) of heavy chain position; Comprising Serine (S) or Threonine (T) on 103 (the AHo numberings) of heavy chain position and/or comprising Serine (S) or Threonine (T) on 144 (the AHo numberings) of heavy chain position.In one embodiment, the framework of identifying based on the choice criteria of selecting can further be modified the modification of assembling to have minimizing of the present invention.For example, if variable light chain framework has arginine (R) through identifying on position 50, so available different amino acid is the locational residue of arginine (R) displacement AHo for example, if perhaps variable heavy chain is through identifying tool Serine (S) on AHo position 12, the residue on so available Threonine displacement AHo position 103 and 144.

As used herein, " humanization " antibody is the antibody that comprises the frame sequence of the variable light chain that variable heavy chain that inhuman CDR and people or people derive and/or people or people derive.The humanization of antibody is well known in the art.In one embodiment, humanized antibody comprises at least 1, preferred 6, comes the CDR of the antibody that produces in comfortable rabbit or is selected from the CDR in CDR library.

can be for example based on identity and/or the similarity of the variable frame sequence of the antibody that is derived from CDR, or based in addition preferred frame sequence, from database (Kabat database for example, Genbank (http://www.ncbi.nlm.nih.gov/genbank/), VBASE (http://vbase.mrc-cpe.cam.ac.uk/), VBASE2 (http://www.vbase2.org/), The Kabat Database of Sequences of Proteins of Immunological Interest (http://www.kabatdatabase.com/index.html), Universal Protein Resource (UniProt, http://pir.georgetown.edu/) and Abysis database (http://www.bioinf.org.uk/abs/)) in select variable antibody framework.

Various computer programs can be used for seeking the suitable people's frame sequence that satisfies selected requirement.For example, " KabatMan " but be the version of searching from the computer of the Kabat antibody sequence data of Sequences of Immunological Interest books.The KabatMan program description is in paper: Martin (1996) Accessing the Kabat Antibody Sequence Database by Computer PROTEINS:Structure, Function and Genetics, 25, in 130-133, and can be available from http://www.bioinf.org.uk/abs/simkab.html and http://www.bioinf.org.uk/abs/kabatman.html.Abysis database (http://www.bioinf.org.uk/abysis/) will be integrated with the structured data from PDB from the sequence data of Kabat, IMGT and PDB.It provides and has allowed by various standard search sequence datas and with the comprehensive interface of namely namely selecting of multi-form demonstration result.For the data from PDB, can sequence search and structure qualification (constraint) is combined.

In yet another aspect, the invention provides the antibody that is produced by method disclosed herein.In preferred embodiments, described antibody comprises having at least 80% identity with SEQ ID NO:12 or SEQ ID NO:13, more preferably 85%, 90%, 95%, 96%, 97%, 98%, and the more preferably VL antibody framework of 99% identity; Preferably, antibody comprises arginine (R) on the AHo position 47 of variable light chain and/or AHo position 50.

In addition or alternatively, VH antibody framework for or comprise SEQ ID NO:3 or have at least 80% identity with SEQ IDNO:3, more preferably 85%, 90%, 95%, 96%, 97%, 98%, the more preferably sequence of 99% identity.

In certain embodiments, the present invention also provides:

(1) be used for reducing it is the method for gathering tendency of antibody that comprises the heterodimer mixture of heavy chain and light chain variable structural domain, and described method comprises step:

(a) provide the complete atomics molecule of antibody to present;

(b) measure the free energy at the interface between two structural domains;

(c) be used for replacing by the following amino-acid residue at one or more participations interfaces of selecting: be provided at the molecular model that comprises the antibody of one or more displacements on selected position, and measure the free energy at the interface that the molecule of displacement presents;

(d) if the interfacial free energy of the molecular model of displacement lower than the interfacial free energy of initial molecular model, selects this amino-acid residue to be used for replacing;

(2) method of (1) wherein in the background of implicit expression solvent method, is measured the free energy at interface by the capacity volume variance between the energy sum of calculating mixture and independent structural domain;

(3) method of (2), wherein said solvent are GBMV or PBSA;

(4) method of any one of aforementioned (1)-(3), it also comprises step:

(i) charge distribution of simulated albumin matter is wherein carried out described step in step a and b;

(5) method of (4) wherein distributes based on electrostatic force or Van der Waals for charge simulation;

(6) method of any one of aforementioned (1)-(5), wherein step (c) is included in the zone of sudden change and carries out the additional step of energy minimization;

(7) method of any one of aforementioned (1)-(7), wherein said antibody are single chain variable fragment (scFv).

Can use the routine techniques in genetic recombination field to produce antibody of the present invention.In the situation that the sequence of known peptide, can utilize method well known in the art to synthesize to produce its cDNA of coding by gene.This type of cDNA clone can be entered suitable vector plasmid.

Can be with well known to a person skilled in the art that standard clone and induced-mutation technique come plus couplings, reorganization (shuffle) structural domain or build fusions to produce the Fab fragment.The base case that discloses general method of the present invention is described in Molecular Cloning, A Laboratory Manual (Sambrook﹠amp; Russell, the 3rd edition .2001) and in Current Protocols in Molecular Biology (people such as Ausubel, 1999).

With the DNA sequence dna (gene with coding scFv polypeptide, or in the situation that the Fab fragment, encode two genes that separate or comprise VL-C κ and two genes of VH-CH1 fusions two along anti-operon) be cloned into suitable expression vector, preferably have the expression vector of inducible promoter.Must be noted that, supply suitable ribosome bind site to guarantee translation in each gene prerequisite.Should be understood that antibody of the present invention comprises disclosed sequence, but not consisting of.For example, Strategies For The Cloning may need to prepare such construct, and it can be provided at the antibody that has one or some extra residues on N-terminal.Especially, the methionine(Met) that produces from initiator codon can be present in (in the situation that it does not excise after being translated) whole protein.Most of constructs of scFv antibody produce other L-Ala on N-terminal.In a preferred embodiment of the invention, select to be used for carrying out intestinal bacteria (E.coli) expression vector (Krebber, 1997) of periplasmic expression.Described carrier comprised promotor before the signal sequence that can cut.Encoding sequence with antibody peptide meets frame ground fusion to the signal sequence that can cut subsequently.This allows the polypeptide targeted bacteria pericentral siphon of expression, and signal sequence is cut in described pericentral siphon.Antibody folds subsequently.In the situation that the Fab fragment must be connected to output signal with VL-C κ and VH-CH1 fusogenic peptide.After peptide arrives pericentral siphon, form covalency S-S key on the C-terminal halfcystine.If the cytoplasmic expression of antibody is preferred, usually can obtain described antibody from inclusion body with high yield, described inclusion body can be easily and other cell fragment and protein separation.In this case, with solubilization of inclusion bodies at denaturing agent for example in Guanidinium hydrochloride (GndHCl), subsequently by well known to a person skilled in the art that refolding method carries out refolding.

To express the plasmid of scFv or Fab polypeptide and introduce suitable host, preferred bacterium, yeast or mammalian cell, most preferably for example JM83 (being used for periplasmic expression) or BL21 (being used for expressing at inclusion body) of suitable coli strain.Can be from pericentral siphon or from inclusion body results polypeptide, and can for example ion exchange chromatography, reversed phase chromatography, affinity chromatography and/or gel-filtration come the described polypeptide of purifying with standard technique well known by persons skilled in the art.

Can characterize antibody of the present invention aspect productive rate, solubleness and vitro stability.For example, can pass through ELISA or surface plasma resonance technology (BIACore), come vitro test to TNF with the recombinant human TNF that describes in WO9729131, preferably to the binding ability of human TNF alpha, a kind of rear method also allows to measure k _offRate constant, they preferably should be less than 10 ^-3s ^-1The K of≤10nM _dValue is preferred.

In one embodiment, the invention provides the α in conjunction with TNF, thereby be adapted at the antibody of the function of blocking-up TNF α in body.In specific embodiment, Anti-tnfa antibody comprises the sequence of SEQ ID NO:10 or SEQ ID NO:17.

Use for therapeutic, use for Mammals (preferred people) with pharmaceutically acceptable formulation (formulation of for example above discussing) anti-TNF antibody of the present invention, described formulation for example comprises, can be by intravenous route (with the form of a notes or pass through continuous infusion within for some time), by the formulation of using to the people in intramuscular, intraperitoneal, myelencephalon, in subcutaneous, intraarticular, synovial membrane, in dura mater, in oral, local, intraocular, nose, through ear, hypogloeeis, through skin or inhalation route.Antibody also can by in tumour, by tumour, around intralesional or focus, approach is suitably used, to produce part and systemic treatment effect.

In order to prevent or treat disease, what suitable antibody dosage will depend on the severity of type (as mentioned in definition), disease of disease to be treated and process, antibody uses preventative or therapeutic purpose, previous therapy, patient's clinical history and the reaction of antagonist, and doctor in charge's judgement.Antibody is suitably used to the patient once or in a series of therapeutic processes.

Anti-TNF antibody of the present invention is useful in the disease for the treatment of TNF mediation.The type and the severity that depend on disease, approximately (for example, antibody 0.1-20mg/kg) is the initial candidate dosage of using to the patient to 1 μ g/kg, no matter for example by independent the using or pass through continuous infusion of one or many to about 50mg/kg.Day commonly used or the scope of weekly dose can be for about 1 μ g/kg to about 20mg/kg or more, and this depends on above-mentioned factor.Repetitive administration within a couple of days or longer time depends on the patient's condition, and repetitive therapy is until the inhibition of the disease symptoms of expectation occurs.Yet other dosage regimen can be useful.The progress of this therapy can easily be monitored by routine techniques and mensuration (for example comprising the radiation oncology imaging technique).

according to another embodiment of the invention, the effect of antibody in prevention or treatment disease can or be that effective agent combination use improve with another kind for this type of purpose with antibody by continuous administration antibody, and described reagent is vascular endothelial growth factor (VEGF) for example, the antibody of angiogenic activity of acidity or Prostatropin (FGF) or pHGF (HGF) can suppress or neutralize, can suppress or in and tissue factor, PROTEIN C, the antibody of the CA of Protein S (referring to people such as Esmon, the open case No.WO91/01753 of the PCT patent of announcing on February 21st, 1991), can be in conjunction with the antibody of HER2 acceptor (referring to people such as Hudziak, the open case No.WO89/06692 of the PCT patent of announcing on July 27th, 1989) or one or more conventional treatment agent alkylating agent for example, antifol, the metabolic antagonist of nucleic acid metabolism, microbiotic, pyrimidine analogue, 5 FU 5 fluorouracil, cis-platinum, purine nucleoside, amine, amino acid, Ribavirina or reflunomide.This type of other reagent can be present in composition to be administered or can use separately.Similarly, continuous administration antibody suitably, or with itself and radiotherapy (no matter comprise irradiation or use radioactive substance) combined administration.

Antibody of the present invention can be used as affinity purification reagent.In the method, use method well known in the art, antibody is fixed on solid phase for example on Sephadex resin or filter paper.With fixing antibody with comprise target protein to be purified (or its fragment) that this antibody is combined (for example in the situation that Anti-tnfa antibody, TNF) contact of sample utilizes subsequently and removes in sample basically that the appropriate solvent of all material (except the target protein that is bonded to fixing antibody) washs upholder.At last, utilize for example glycine buffer of the another kind of appropriate solvent that will discharge target protein from antibody, pH5.0 washs upholder.

Antibody also can be used for the diagnostic assay of target protein, for example detects its expression in specific cells, tissue or serum.This type of diagnostic method can be used for cancer diagnosis.

For diagnostic use, usually use detectable part traget antibody.Many markers are obtainable, and it can be divided into following classification usually:

(a) radio isotope, for example ¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, ³H, ³²P or ³⁵S.Can use for example Current Protocols in Immunology, the 1st and 2 volumes, the people such as Coligen, Ed.Wiley-Interscience, New York, the technology of describing in N.Y., Pubs. (1991), use labelled with radioisotope antibody, and can measure radioactivity with scintillation counting technique.

(b) for example rare earth sequestrant (europium sequestrant) or fluorescein and derivative, rhodamine and derivative thereof, dansyl, Liz amine, phycoerythrin and texas Red are obtainable to fluorescent marker.For example can use in Current Protocols in Immunology (the same) disclosed technology that fluorescent marker is puted together in antibody.Can use the photofluorometer quantitative fluorescence.

(c) various enzyme-substrate markers are obtainable, and U.S. Patent No. 4,275,149 provides the summary of some these type of markers.The chemically changed of the common catalysis chromogenic substrate of enzyme, this can measure with various technology.For example, but the colour-change of enzyme catalytic substrate, and this can utilize spectrophotometer to measure.Perhaps, enzyme can change fluorescence or the chemoluminescence of substrate.The technology that quantitative fluorescence changes that is used for has above been described.Chemical luminous substrate can be by the chemical reaction electron excitation, can launch subsequently light that can measured (for example using chemoluminescence meter (chemiluminometer)) or energy is offered fluorescent receptor.The example of enzymatic labelling thing comprises luciferase (for example, Fluc and bacteriofluorescein enzyme; U.S. Patent No. 4,737,456), luciferin, 2,3-dihydro phthalazine diketone, malate dehydrogenase (malic acid dehydrogenase), urease, peroxidase be horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase enzymes, glucoamylase, N,O-Diacetylmuramidase, carbohydrate oxidase (for example, notatin, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocycle oxydase (such as uriKoxidase and XOD), lactoperoxidase, microperoxisome etc. for example.Be used for enzyme is conjugated to the technical description of antibody in people such as O'Sullivan, Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (ed J.Langone﹠amp; H.Van Vunakis), Academic press, New York is in 73:147-166 (1981).The example of enzyme-substrate combination for example comprises:

(i) horseradish peroxidase (HRPO) with as the catalase of substrate, wherein catalase oxidation dye precursors (for example, former phenylene diamines (OPD) or 3,3', 5,5'-tetramethyl biphenyl amine hydrochlorate (TMB));

(ii) alkaline phosphatase (AP) with as the p-nitrophenyl phosphoric acid of chromogenic substrate; With

(iii) beta-D-galactosidase (β-D-Gal) and chromogenic substrate (for example, p-nitrophenyl-beta-D-galactosidase) or fluorogenic substrate 4-methyl umbelliferone-beta-D-galactosidase.

Many other enzyme-substrate combinations are obtainable to those skilled in the art.About the general summary of this type of combination, referring to U.S. Patent No. 4,275,149 and 4,318,980.Sometimes, marker and antibody indirect are puted together.Those skilled in the art know, and are used for realizing the various technology that this is puted together.For example, can be with antibody and biotin-conjugated, and any and avidin of the marker of above-mentioned 3 wide class can be puted together, or vice versa.Vitamin H selective binding avidin, thus can marker and antibody be puted together with this indirect mode.Perhaps, in order to realize indirectly puting together of marker and antibody, antibody and little haptens (for example, digoxin) to be puted together, and one of above-mentioned dissimilar marker and anti--hapten antibody (for example, anti--DigiTAb) are puted together.Thus, can realize indirectly puting together of marker and antibody.

In certain embodiments, needn't traget antibody, and its existence can detect with the antibody through mark in conjunction with target antibody.

Antibody of the present invention can be used for any known measuring method, for example in competitive binding assay, direct and indirect sandwich assay and immune precipitation determination.Zola,Monoclonal?Antibodies:A?Manual?of?Techniques,pp.147-158(CRC?Press,Inc.1987)。

Competitive binding assay depends on through the ability of the standard substance of mark and specimen analyte competition to the combination of limited amount antibody.For example, the amount of the TNF albumen in specimen is inversely proportional to the amount that is bonded to the standard substance of antibody.For the amount of the standard substance that helps to measure combination, antibody is normally insoluble before or after competition, thereby can separate with analyte with the unconjugated standard substance of analyte and maintenance in connection with the standard substance to antibody easily.

Sandwich assay comprises the use of two kinds of antibody, and each antibody can be in conjunction with different immunogenicity parts or the epi-position of protein to be detected.In sandwich assay, the specimen analyte is fixed on the first antibody combination on solid support, second antibody bound analyte subsequently, thus form insoluble three part mixtures.Referring to, for example, U.S. Patent No. 4,376,110.The available detectable part mark of second antibody itself (directly sandwich assay) but but or applying marking have the test section anti--immune globulin antibody measures (sandwich assay indirectly).For example, a type of sandwich assay is that ELISA measures, and detectable part is enzyme in this case.

For immunohistochemistry, tissue sample for example tumor sample can be fresh or freezing, maybe can be embedded in paraffin and with sanitas for example formalin be fixed.

Antibody also can be used for the in-vivo tumour diagnostic assay.Usually, antibody can be with radionuclide (for example ¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, ³H, ³²P or ³⁵S) carry out mark, in order to can use immune scintigraphy (immunoscintiography) to carry out positioning tumor.

Can provide antibody of the present invention at test kit the combination through packing of the specification sheets that is used for carrying out diagnostic assay (reagent of predetermined amount with).When using enzymic-labelled antibody, test kit will comprise substrate and the cofactor (the substrate precursor of detectable chromophore or fluorophore for example, is provided) that enzyme is required.In addition, can comprise other additive for example stablizer, buffer reagent (for example, sealing buffer reagent or cracking buffer reagent) etc.The relative quantity of all ingredients can change widely, so that the concentration of optimizing significantly the susceptibility of measuring to be provided in the solution of reagent.Especially, can provide reagent with the dry powder form (commonly lyophilised) that comprises vehicle, it will provide the reagent solution with proper concn after dissolving.

The present invention also provides the pharmaceutical preparation that comprises one or more antibody of the present invention that are used for the treatment of purpose.In one embodiment, the invention provides the anti-TNF antibody of the disease that is used for the treatment of the TNF mediation.

Term " pharmaceutical preparation " refers to, with the preparation that such form exists, its biologic activity that makes antibody is effectively clear and definite, and does not comprise the other component poisonous to the experimenter who uses described preparation to it." pharmaceutically acceptable " vehicle (vehicle, additive) can reasonably be administered to the vehicle of tested Mammals with the activeconstituents that is used that effective dose is provided.

" stable " preparation is that wherein herein antibody keeps the preparation of its physical stability and/or chemical stability and/or biologic activity basically when storing.The various analytical technologies that are used for the measurement protein stability are obtainable in the art and summarize in for example Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones are in A.Adv.Drug Delivery Rev.10:29-90 (1993).Can be at the temperature of selecting measurement stability, the time period of selecting.Preferably, preparation was under room temperature (approximately 30 ℃) or 40 ° of C stable at least 1 month and/or at approximately under 2-8 ℃ stable at least 1 year or at least 2 years.In addition, preparation is preferably freezing (to for example ,-70 ℃) of preparation be stable after thawing.

If antibody after the visual inspection of color and/or clarity, or as by UV-light scattering or measure by size exclusion chromatography, does not show the sign of gathering, precipitation and/or sex change, it " keeps its physical stability " in pharmaceutical preparation.

If make protein be considered to still keep as undefined its biologic activity in given temporal chemical stability, antibody " keeps its chemical stability " in pharmaceutical preparation.Chemical stability can be by detecting and the form of the chemically changed of quantitative protein is assessed.Chemically changed for example can comprise, can use the size of size exclusion chromatography, SDS-PAGE and/or substance assistant laser desorpted ionized flight time mass spectrum (MALDI/TOF MS) assessment to modify (for example, shearing (clipping)).The chemically changed of other type for example comprises, can change by the electric charge that ion exchange chromatography is estimated (electric charge that for example, occurs due to deacylated tRNA amine changes).

If in (in the error of measuring) in approximately 10% error of the biologic activity that the biologic activity of antibody on the given time is showed when for example pharmaceutical preparation is produced, as at antigen in conjunction with measuring in measuring, antibody " keeps its biologic activity " in pharmaceutical preparation.Other of antibody " biologic activity " is determined at and hereinafter carries out detailed description.

" wait and ooze " to mean, target formulation has the osmotic pressure substantially the same with human blood.Usually has approximately 250 to 350mOsm osmotic pressure Deng oozing preparation.Can use the permeameter of vapour pressure for example or freezing type to measure isotonicity.

" polyvalent alcohol " comprises sugar (reductibility and nonreducing sugar), sugar alcohol and saccharic acid for having the material of a plurality of hydroxyls.Preferred polyol herein has the molecular weight lower than about 600kD (for example approximately 120 to about 400kD scope in)." reducing sugar " be have the reducible metal ion or with protein in Methionin and the sugar of the hemiacetal group of other amino covalence reaction, and " nonreducing sugar " is the sugar with this class character of reducing sugar.The example of reducing sugar is fructose, seminose, maltose, lactose, pectinose, wood sugar, ribose, rhamnosyl, semi-lactosi and glucose.Nonreducing sugar comprises sucrose, trehalose, sorbose, melizitose and raffinose.N.F,USP MANNITOL, Xylitol, erythritol (erythritol), threitol, sorbyl alcohol and glycerine are the examples of sugar alcohol.About saccharic acid, it comprises L-glyconic acid and metal-salt thereof.When the expectation preparation is freeze-thaw when stable, polyvalent alcohol is preferably at the polyvalent alcohol of freezing temperature (for example-20 ℃) lower non-crystallizable (so that its antibody in making preparation goes to stablize).Nonreducing sugar includes but not limited to, sucrose and trehalose.

As used herein, " buffer reagent " refers to, resists the buffered soln of the change of pH by the effect of its acidity-alkaline conjugation component.Buffer reagent of the present invention has approximately 4.5 to approximately 7.0; Preferred approximately 4.8 to about 6.5 pH.The example that pH can be controlled at the buffer reagent in this scope comprises acetate (for example, sodium-acetate), succinate (for example sodium succinate), gluconate, Histidine, Citrate trianion and other organic acid buffer reagent.When wanting the freeze-thaw stability preparation, buffer reagent is not preferably phosphoric acid salt.

On pharmacological significance, in background of the present invention, " the treatment significant quantity " of antibody refer to, is effective amount in the prevention of effective illness or treatment for its treatment at antibody." disease/illness " is any patient's condition that can benefit from the treatment that utilizes antibody.This comprises chronic and acute disease or disease, comprises making Mammals easily suffer from those pathology patient's condition of described illness.

" sanitas " is for example to be included in preparation to reduce substantially bacteriological action wherein, thereby is conducive to the compound of the generation of multi-usage preparation.The example of potential sanitas comprises, octadecyl dimethyl benzyl ammonium chloride, Meton, benzalkonium chloride (wherein alkyl is the mixture of the chlorination alkyl benzyl dimethyl ammonium of long-chain compound) and benzethonium chloride.The sanitas of other type comprises for example for example methyl p-hydroxybenzoate or propyl ester, catechol, Resorcinol, hexalin, 3-amylalcohol and m-cresol of phenol, butyl and benzyl alcohol, alkyl paraben of aromatic alcohol.Most preferred sanitas herein is benzyl alcohol.

The present invention also provides the pharmaceutical composition that comprises one or more antibody and at least a physiologically acceptable carrier or vehicle.Pharmaceutical composition (for example for example can comprise water, buffer reagent, neutral buffered salts solution or phosphate buffered saline(PBS)), ethanol, mineral oil, vegetables oil, methyl-sulphoxide, carbohydrate (for example, glucose, seminose, sucrose or dextran), N.F,USP MANNITOL, protein, adjuvant, polypeptide or amino acid glycine, antioxidant, sequestrant one or more in EDTA or gsh and/or sanitas for example for example.Pointed as mentioned, can (but not essential) comprise other activeconstituents in the pharmaceutical composition that provides in this article.

Carrier is can be before using to the patient and the material of antibodies (being generally used for controlling stability or the bioavailability of compound).The carrier that uses in this type of preparation is biocompatibility normally, and can also be biodegradable.Carrier for example comprises for example for example glycosaminoglycan and daiamid of serum albumin (for example, people or ox), Protalbinic acid, peptide, poly-lysine and polysaccharide of unit price or multivalent molecule.Carrier also comprises solid support material for example bead and particulate, and it comprises for example poly(lactic acid), polyglycolic acid, poly-(lactic acid/oxyacetic acid) multipolymer, polyacrylic ester, latex, starch, Mierocrystalline cellulose or dextran.Carrier can be in many ways (comprising covalent bonding (directly or by the joint group), noncovalent interaction or mixing) carries compound.

But the compounding pharmaceutical composition to be to be used for any suitable method of application, for example comprises in eye, nose, through ear, hypogloeeis, use through skin, part, oral, intranasal, per rectum or parenteral.In certain embodiments, the composition that exists with the form that is applicable to orally use is preferred.This type of form comprises for example pill, tablet, lozenge (troches), lozenge, water-based or oily suspensions, dispersible pulvis or particle, emulsion, hard or soft capsule or syrup or elixir.In other embodiments, the composition that provides herein can be formulated as lyophilized products.As used herein, the term parenteral comprise (for example, intravenously), intramuscular in subcutaneous, intradermal, blood vessel, in backbone, encephalic, dura mater and peritoneal injection and any similar injection or infusion techniques.

In certain embodiments, antibody of the present invention can for example be injected near the eyes by ocular tissue, injection directly is delivered to eye under conjunctiva, ball manadesma, in the anterior chamber, in vitreum, under intraocular, retina, under conjunctiva, after eyeball or in tubule; For example retina tablet (retinal pellet), intraocular inset, suppository or the implant that comprises porous, atresia or gel-like material are applied directly to eye by using conduit or other arranging device; By topical eye drops or ointment; By in pouch (cul-de-sac) or contiguous sclera (trans-scleral) or in sclera (intrascleral) or directly be delivered to eye in the delayed release device of Vitreous cavity.Intracameral injection can enter the anterior chamber via cornea, arrives trabecular network (trabecular meshwork) to allow reagent.In tubule, injection can enter the vein collecting tubule of drainage Schlemm's canal (Schlemm's canal) or enter Schlemm's canal.

Send for eye, can be with antibody of the present invention and the acceptable sanitas of ophthalmology, cosolvent, tensio-active agent, viscosity-increasing agent, penetration enhancers, buffer reagent, sodium-chlor or water combination, to form the aseptic ophthalmic suspension of water-based or solution.Can be for example with multiple doses packaged topical ophthalmic product.Therefore, may need sanitas in use to prevent microbial contamination.Suitable sanitas comprises: trichlorine uncle's butanone, methyl p-hydroxybenzoate, propylparaben, styroyl alcohol, Zonon D, Sorbic Acid, polyquaternium-1 or other reagent well known by persons skilled in the art.This type of sanitas uses with 0.001 to 1.0%w/v level usually.Units dosage composition of the present invention is aseptic, and (unpreserved) of preservative-free normally.Such composition thereby usually do not comprise sanitas.

In certain embodiments, will be intended to topical application to the composition of eye and be formulated as eye drops or Eye ointments, wherein the total amount of antibody is about 0.001 to 1.0% (w/w).Preferably, the amount of TNF Alpha antibodies is approximately 0.01 to about 1.0% (w/w).

In some cases, composition of the present invention is used as the solution that is used for topical application.Based on the ability that easiness and the patient of preparation easily uses such composition (by 1 to 2 solution being splashed in ill eye), the aqueous solution is normally preferred.Yet composition can be also suspension, thickness or half viscous gel, or the solid of other type or semi-solid combination.

Be intended to prepare according to any method for the manufacture of pharmaceutical composition known in the art be used to the composition that orally uses, and it can comprise one or more reagent, for example sweeting agent, seasonings, tinting material and sanitas are to provide attracting and good to eat preparation.Tablet comprises the activeconstituents with the physiologically acceptable mixed with excipients that is suitable for making tablet.This type of vehicle comprises that for example inert diluent (for example; calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate), granulating agent and disintegrating agent (for example; W-Gum or Lalgine), tackiness agent (for example; starch, gelatin or or gum arabic (acacia)) and lubricant (for example, Magnesium Stearate, stearic acid or talcum).Tablet can maybe can utilize known technology to be coated with without dressing, with delay disintegration and absorption in gi tract, thereby provides continuous action within longer period.For example, the serviceable time postpones material for example glyceryl monostearate or distearin.

Being used for the preparation orally use can also be with the form of hard gelatin capsule (wherein with activeconstituents and inert solid diluent (for example, calcium carbonate, calcium phosphate or kaolin) mix) or provide with the form (wherein activeconstituents and water or oily medium (for example, peanut oil, whiteruss or sweet oil) being mixed) of soft gelatin capsule.Aq suspension comprises the antibody with the mixed with excipients that is suitable for making aq suspension.This type of vehicle comprises suspensoid (for example, Xylo-Mucine, methylcellulose gum, Vltra tears, sodium alginate, polyvinylpyrrolidone, tragacanth gum and gum arabic); With dispersion agent or wetting agent (for example, naturally occurring phosphatide for example Yelkin TTS, alkene oxide and lipid acid condensation product for example polyoxyethylene 8 stearate, oxyethane and long chain aliphatic alcohol condensation product for example heptadecaethyleneoxycetanol, oxyethane and from the condensation product of lipid acid and the derivative partial ester of the hexitol condensation product polyethylene sorbitan monooleate for example of octadecanoic acid ester of polyethylene glycol or oxyethane and the partial ester that derives from lipid acid and hexitan for example).The aqueous suspension agent also can comprise one or more sanitass, for example ethyl p-hydroxybenzoate or just-propyl ester, and one or more tinting materials, one or more seasoningss and one or more sweeting agents be sucrose or asccharin for example.Can utilize sweeting agent for example glycerine, propylene glycol, sorbyl alcohol or the agent of sucrose obtain syrup and elixir.This type of preparation also can comprise one or more negative catalyst (demulcent), sanitas, seasonings and/or tinting material.

Oily suspensions can for example be prepared in whiteruss by activeconstituents being suspended in vegetables oil (for example, peanut oil, sweet oil, sesame oil or Oleum Cocois) or mineral oil.Oily suspensions can comprise thickening material for example beeswax, paraffinum durum or hexadecanol.Can add sweeting agent (for example above described sweeting agent) and/or seasonings so that agreeable to the taste oral preparations to be provided.This type of suspension can by add antioxidant for example xitix preserve.

The dispersible pulvis or the granule that are suitable for preparing by adding water waterborne suspension provide the activeconstituents that mixes with dispersion agent or wetting agent, suspensoid and one or more sanitass.Those that suitable dispersion agent or wetting agent and suspensoid are for example mentioned hereinbefore.Also can there be other vehicle for example sweeting agent, seasonings and tinting material.

Pharmaceutical composition can also exist with the form of oil-in-water emulsion.Oil phase can be vegetables oil (for example, sweet oil or peanut oil), mineral oil (for example, whiteruss) or its mixture.Suitable emulsifying agent (for example comprises naturally occurring natural gum, gum arabic or tragacanth gum), naturally occurring phosphatide (for example, soybean lecithin and from lipid acid and hexitol derivative ester or partial ester), acid anhydride (for example, sorbitan monooleate) and from the derivative partial ester of lipid acid and hexitol and the condensation product (for example, polyoxyethylene sorbitan monooleate) of oxyethane.Emulsion also can comprise one or more sweeting agents and/or seasonings.

Pharmaceutical composition can be prepared as sterile injectable water-based or oily suspensions, wherein depend on vehicle and the concentration used, conditioning agent is suspended or is dissolved in vehicle.Can be according to known technology, those prepare such composition as mentioned in the text with suitable dispersion agent, wetting agent and/or suspensoid example.Spendable acceptable vehicle and solvent are water, 1,3 butylene glycol, Ringer's solution and isotonic sodium chlorrde solution.In addition, aseptic fixed oil can be can be used as solvent or suspension medium.For this purpose, the fixed oil of any gentleness be can use, synthetic monoglyceride or triglyceride comprised.In addition, lipid acid for example oleic acid can be used for preparing Injectable composition, and can for example local anesthetic, sanitas and/or buffer reagent are dissolved in vehicle with adjuvant.

Pharmaceutical composition can be formulated as extended release preparation (that is, realizing formulation example such as the capsule of the slow release of conditioning agent after using).This type of preparation can prepare and can be by for example oral, per rectum or subcutaneous implantation with known technology usually, or by implanting to use at the target position of expectation.The carrier that uses in this type of preparation is biocompatibility, and can be also biodegradable; Preferably, preparation provides the conditioning agent of relative constant level to discharge.The amount of the antibody that comprises in extended release preparation for example depends on the character of the disease/illness of time length of the speed of the position of implanting, release and expection and to be treated or prevention.

Can with reach in body fluid (for example, blood, blood plasma, serum, CSF, synovia, lymph liquid, intercellular washing fluids, tear or urine) be enough to can detect use in conjunction with TNF and prevention or the amount of concentration that suppresses the disease/illness of TNF mediation the Anti-tnfa antibody that provides herein.As described in this article, if dosage causes recognizable patient's benefit, think that this dosage is effective.Preferred systemic doses scope is that approximately 0.1mg is to the about body weight/day of 140mg/ kilogram (approximately 0.5mg to approximately 7g/ patient/day), and oral dosage is generally approximately 5-20 times of intravenous dosages.Can will depend on host to be treated and concrete mode of administration with the amount that solid support material makes up to produce the antibody of single dosage form.Dosage unit form can comprise approximately 1mg usually to the about activeconstituents of 500mg.

In certain embodiments, but the packaged pharmaceuticals composition to be used for the treatment of the patient's condition that anti-TNF antibodies is reacted.The pharmaceutical composition of packing (for example can comprise the container of at least a antibody described herein that loads significant quantity and specification sheets, labeling), described specification sheets indicates, the composition that comprises is used for the treatment of disease/illness that described antibody is reacted after using to the patient.

But chemically modified antibody of the present invention also.Preferred modification group is polymkeric substance, for example randomly the straight or branched polyolefine (polyalkene) of replacement, poly-alkenylene (polyalkenylene) or polyoxygenated alkene (polyoxyalkylene) polymkeric substance or branch or the not polysaccharide of branch.This type of effector group can increase the Half-life in vivo of antibody.The specific examples of synthetic polymkeric substance comprises the straight or branched PEG (PEG) that randomly replaces, poly-(propylene glycol), poly-(vinyl alcohol) or derivatives thereof.Concrete naturally occurring polymkeric substance comprises lactose, amylose starch, dextran, glycogen or derivatives thereof.Can change as required the size of polymkeric substance, but its size is usually in the average molecular weight range of 500Da to 50000Da.Be designed to permeate the topical application of tissue for antibody wherein, the preferred molecular weight of polymkeric substance is about 5000Da.Polymer molecule can be connected in antibody, for example be connected in the C-terminal of Fab fragment heavy chain by the covalently bound hinge peptide of describing in WO0194585.Connection about peg moiety, with reference to " Poly (ethyleneglycol) Chemistry; Biotechnological and Biomedical Applications ", 1992, J.Milton Harris (ed), Plenum Press, New York and " Bioconjugation Protein Coupling Techniques for the Biomedical Sciences ", 1998, M.Aslam and A.Dent, Grove Publishers, New York.

After the above-mentioned target antibody of preparation, preparation comprises its pharmaceutical preparation.Antibody to be prepared does not experience formerly freeze-drying and target formulation herein is aqueous compositions.Preferably, the antibody in preparation is for example scFv of antibody fragment.The treatment significant quantity that dose volume by considering for example expectation and mode of administration determine to be present in the antibody in preparation.Approximately 0.1mg/ml is to about 50mg/ml, preferred approximately 0.5mg/ml to about 25mg/ml and most preferably from about 2mg/ml extremely approximately 10mg/ml be exemplary antibodies concentration in preparation.

Preparation comprises the aqueous compositions of antibody in above-mentioned pH buffered soln.Depend on for example isotonicity of the expectation of buffer reagent and preparation, buffer concentration can be approximately 1mM to about 50mM, and preferred approximately 5mM is to about 30mM.

Can comprise in preparation as tonicity agents (tonicifier) but and the polyvalent alcohol of stabilization of antibodies.In preferred embodiments, the salt that preparation does not comprise tension force (tonicifying) amount is sodium-chlor for example, because this can cause the antibody precipitation and/or can cause oxidation under low pH.In preferred embodiments, polyvalent alcohol is for example sucrose or trehalose of nonreducing sugar.With the amount that can change with the preparation isotonicity of expectation, polyvalent alcohol is added into preparation.Preferably, aqueous compositions etc. ooze, in this case in preparation the proper concn of polyvalent alcohol for example approximately in 1% to about 15%w/v scope, preferably in about 2% to about 10%w/v scope.Yet height oozes or hypotonic preparation also can be fit to.The amount of the polyvalent alcohol that adds also can change with the molecular weight of polyvalent alcohol.For example, compare with disaccharides (for example trehalose), can add the monose (for example, N.F,USP MANNITOL) of low amount.

Also tensio-active agent can be added in antibody preparation.Exemplary surfactants comprises nonionic surface active agent for example polysorbate (for example, Polysorbate 20,80 etc.) or poloxamer (for example, PLURONICS F87).Usually, the amount of the tensio-active agent of interpolation makes the gathering of its antibody/antibody derivatives that reduces preparation and/or minimizes the formation of particle in preparation and/or reduce absorption.For example, tensio-active agent can be with approximately 0.001% to approximately 0.5%, and is preferred approximately 0.005% to approximately 0.2% and most preferably from about 0.01% being present in preparation to about 0.1% amount.

In one embodiment, preparation comprises the reagent (being antibody, buffer reagent, polyvalent alcohol and tensio-active agent) of above describing and is substantially free of one or more sanitass, for example phenylcarbinol, phenol, m-cresol, trichloro-butyl alcohol and benzethonium chloride (benzethonium Cl).In another embodiment, can comprise sanitas in preparation, especially when preparation is multi-dose formulation.The concentration of sanitas can be approximately 0.1% to approximately 2%, most preferably from about 0.5% to approximately 1%.Can comprise for example Remington's Pharmaceutical Sciences of one or more other pharmaceutically acceptable carrier, vehicle or stablizer in preparation, the 21st edition, Osol, A.Ed. those that describe in (2006) are as long as they can not adversely affect the formulation characteristics of expectation.Acceptable carrier, vehicle or stablizer are nontoxic to the receptor on the dosage that uses and concentration and comprise: other buffer reagent; Cosolvent; Antioxidant comprises xitix and methionine(Met); Sequestrant is EDTA for example; Metal composite (for example, Zn-albumen composition); Biodegradable polymeric is polyester for example; And/or salt formation counter ion sodium for example.

The preparation that is used for using in body must be aseptic.This can be easily by filtering to realize via sterile filtration membrane before or after the preparation preparation.

For example intravenously is used (with the form of a notes or by the continuous infusion within for some time) in accordance with known methods, by in intramuscular, intraperitoneal, myelencephalon, in subcutaneous, intraarticular, synovial membrane, in dura mater, oral, part or inhalation route, or other approach of describing herein, utilize the preferred people's administered formulation of Mammals of the treatment of antibody to needs.In certain embodiments, be administered to the administration preparation by intravenously.For this purpose, can for example use syringe or by IV line injection formulations.

The suitable dosage of antibody (" treatment significant quantity ") will depend on the severity of the patient's condition for example to be treated, the patient's condition and process, antibody are preventative or therapeutic administration, previous therapy, patient's clinical history and the reaction of antagonist, the Antibody types that uses and doctor in charge's judgement.Give suitably administration of antibodies of patient once or in a series of therapeutic processes, and any time that can be after diagnosis is to patient's administration of antibodies.Antibody can with the form of single therapy use or be that useful other medicines or therapy is co-administered in the described patient's condition in treatment.

As general recommendations, the treatment significant quantity of the antibody of using will be approximately 0.1 to the scope of about 100mg/kg weight in patients (no matter being once or repeatedly to use), the common scope of the antibody that uses of for example using every day is approximately 0.3 to about 20mg/kg, more preferably from about 0.3 to about 15mg/kg.Yet other dosage can be useful.The progress of this therapy can easily be monitored by routine techniques.

In certain embodiments, use the pharmaceutical composition that comprises Anti-tnfa antibody of the present invention for the patient of the illness of suffering from the TNF mediation.

In another embodiment of the invention, goods are provided, described goods comprise the container that loads aqueous medicament preparations of the present invention, and its working instructions randomly are provided.Suitable container comprises for example bottle, bottle and syringe.Can for example glass or plastics form container from multiple material.Exemplary containers is the disposable vial of 3-20cc.Perhaps, for multi-dose formulation, container can be the vial of 3-100cc.Container loads preparation, and on container or the labeling that accompanies with it can show operation instruction.Goods can comprise that also from business and User Perspective be other material of expectation, comprise other buffer reagent, thinner, strainer, pin, syringe and the unit packing list with operation instruction.

The content of any patent, patent application and the bibliography that will quote in whole specification sheets integral body is by reference incorporated this paper into.

Unless the other requirement of context, singular references used herein should comprise plural number, and plural term should comprise odd number.

Embodiment

Further illustrate present disclosure by the following example, described embodiment should not be interpreted as determinate.The content of the patent application of all figure that quote in whole the application and all bibliographys, patent and announcement clearly by reference integral body incorporate this paper into.

In all embodiments, except as otherwise noted, otherwise use following materials and methods.

General materials and methods

Generally speaking, except as otherwise noted, otherwise the standard technique of chemistry, molecular biology, recombinant DNA technology, immunology (especially, for example antibody technique) and polypeptide preparation is used in enforcement of the present invention.Referring to, for example, Sambrook, Fritsch and Maniatis, Molecular Cloning:Cold Spring Harbor Laboratory Press (1989); Antibody Engineering Protocols (Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996); Antibody Engineering:A Practical Approach (Practical Approach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies:A Laboratory Manual, the people such as Harlow, C.S.H.L.Press, Pub. (1999); With Current Protocols in Molecular Biology, the people such as eds.Ausubel, John Wiley﹠amp; Sons (1992).

Thermostability is measured

Use FT-IR Bio-ATR cell to obtain attenuated total reflectance attenuated total refraction Fourier Tranform IR (FTIR-ATR) spectrum and the tracing observation molecule of various strands in Tensor Bruker.Molecule is concentrated into up to 3mg/ml, subsequently under 4 ℃ for PBS, the pH6.5 dialysed overnight is collected the damping fluid flow through as blank.Obtain the Vertic features spectrum by attacking molecule with temperature (stride is 5 ℃, 25 to the 95 ℃) heat of wide region.Use OPUS software to carry out all spectrum operations.Deduction host buffer liquid and transient state atmosphere (CO from protein spectrum ₂And H ₂O) background.Subsequently the protein spectrum of gained carried out the baseline correction, and according to the width measurements protein acid amides I spectrum of wide resolved peak in expected areas.Use cubic polynomial function and smooth function to obtain the second derivative spectra of acid amides I band spectrum.Use the linear calibration curve of virgin curve-the Fitting Calculation, estimate the variation (for 3 lower observed values, suppose 0% sex change, and for 3 higher observed values, suppose 100% sex change) of protein structure by acid amides I derivative analysis of second.Use Boltzmann S curve model, the Vertic features spectrum is used for the mid point of the folding conversion of pyrolysis (thermal unfolding transition, TM) of each variant of estimation.

Solubleness is measured

In the situation that ammonium sulfate exists, assemble and the rear relative solubility of measuring various scFv molecules of precipitation in Enhancin matter.Ammonium sulfate is added in protein water soln, to produce the saturation ratio of increment as 5% in whole mixture salt-protein.Measure by rule of thumb the precipitation in dynamicrange, and in whole mixture, the saturation ratio interval is decreased to 2.5% interval saturation ratio in this scope.After adding ammonium sulfate, biased sample lightly, and with 6000rpm centrifugal 30 minutes.For each ammonium sulfate saturation ratio percentage ratio, reclaim remaining protein in supernatant liquor.By measuring solubility curve with the protein concn in NanoDropTM1000 spectrophotometer measurement supernatant liquor.With the observed value stdn of remaining soluble protein in supernatant liquor, then use it for the mid point of the relative solubility of estimating each variant by using Boltzmann S curve model.

The short-term stability property testing

Check solubility gathering thing and the degraded product of protein after 2 weeks at 40 ℃ of lower incubations.It is the protein of the 10mg/ml PBS dialysed overnight for wide pH scope (3.5,4.5,5.5,6.5,7.0,7.5 and 8.5) under 4 ℃ with concentration.The control protein with same concentrations in standard buffer solution PBS (pH6.5) was stored for 2 weeks under-80 ℃.Measure the degraded band by SDS-PAGE on t=0 and t=14d time point, and estimate solubility and assemble thing in SEC-HPLC.After carrying out for 2 weeks under 40 ℃, use Biacore to measure remaining activity.

Embodiment 1

Optimizing anti-TNF alpha scFv antibody 34rFW1.4 assembles to reduce

34rFW1.4 antibody shows the tendency that the pH dependency is assembled in solution.Do not show the gathering tendency with the 578rFW1.4 antibody (referring to International Application No. WO 2009155724) of the total same architecture structure of 34rFW1.4.Following generation homology model is to identify that can be modified to reduce it in 34rFW1.4 assembles the potential residue that is inclined to.

The variable domains sequence is used for building the homology model of 34rFW1.4 antibody and 578rFW1.4 antibody.In order to produce described model, use based on the search of BLAST algorithm and identify respectively the light chain (VL) of each antibody and the formwork structure of heavy chain (VH) variable domains sequence.Due to the high conservative property of antibody framework region, BLOSUM80 (being used for the right matrix of less diversity ratio) is used as the matrix for comparison.Select the template separately of each chain (VL/VH), itself and search sequence have the identity over 70%.

Based on the variable domains template of identifying, with the program MODELER (Accelrys, Inc., San Diego, CA) of Discovery Studio2.5.5 version (DS2.5.5) software for generation of 100 scFv models.The comparing as the input data with reference to antibody sequence and formwork structure of modeling will be treated.Produce 100 models that comprise all non-hydrogen atoms.Based on the scoring of PDF (probability density function) physical energy, select the best model structure.The relative direction of VH and VL structural domain is set, with the direction of coupling from variable domains formwork structure (two (VL and VH) sequences of itself and modeling have highest homology).Remove the optional conformation (alternate conformation) in model, add end, and add the side chain atom of losing.The scFv model is applied the CHARMM field of force, and use 0.01 RMS gradient and Generalized Born with Molecular Volume (GBMV) (as implicit expression solvent model), carry out 2000 energy minimizations of taking turns.

The protein ionization and the residue pK that carry out each antibody calculate.Based on by Bashford and Karplus, the theories of 1991 exploitations, the actualizing (protocol implementation) that uses Discovery Studio2.5.5 version (DS2.5.5) to comprise calculates.PK according to the side chain of antibody _1/2With each titratable residue titration curve of (comprising Asp, Glu, Arg, Lys, His, Tyr, Cys, N-terminal and C-terminal residue), the result that relatively protein of two molecules ionizes and residue pK calculates.

The scFv model is applied the CHARMM field of force, carry out protonatedly under pH7.4, and use 0.2 pH stride to calculate titration curve and the pK of each residue in the scope of pH2 to pH14.578rFW1.4 two conservative Lys residues on the position 47 and 50 in the light chain of antibody are compared with these the locational Lys in 34rFW1.4, have different (referring to Fig. 1).

The introducing of preferred displacement

Discovery Studio software is used for carrying out the molecule dynamic simulation, improves interaction avidity between VL and VH with prediction, thereby stop structural domain to separate and form the sudden change of oligomer and/or high grade collecting thing.Be energy terms such as free energy G with the stability assessment at the VL/VH interface of 34rFW1.4 antibody.The CHARMM agreement connects (protocol adaptation) (being also included within DS2.5.5) for the capacity volume variance between the energy sum of calculating complete scFv heterodimer mixture and the variable domains that each is independent.Use Generalized Born with Molecular Volume integration (GBMV) to calculate in the background of implicit expression solvent method.Calculate the energy of 2 structural domains and mixture, according to following formula, Output rusults is determined as capacity volume variance between mixture and independent structural domain sum: G interface=G (a)-G (b)-G (c), wherein G (a) is the energy of antibody, G (b) is the energy of VL, and G (c) is the energy of VH.

Arginine (R) is selected to replace as the possible residue of the Methionin on position 47 and 50 (K), because the side chain pKa value (12.5) of R is higher than the pKa (10.5) of K.By producing mutant K47R and K50R with the alternative K of R respectively on the position separately.Adjust the distance sudden change peripheral region 10 dusts or nearer residue carries out 2000 and takes turns energy minimization, so that the New model molecule can Adaptive change.The implicit expression solvent model that is used for the energy minimization of these rounds is GBMV.The average delta G of the prediction of mutant is-94kcal/mol.Therefore, compare with parental generation anti-TNF scFv antibody, the contribution of sudden change is estimated as 4kcal/mol.

Embodiment 2

The stability analysis of the 34rFW1.4 antibody of optimizing

Generation K50R mutant and K47R mutant in 34rFW1.4 antibody (its be disclosed in the common unsettled international application No.PCT/CH2009/000219 that submitted on June 25th, 2009 in, the content of described application integral body is by reference incorporated this paper into).According to U.S. Patent application No.12/973, the method preparation of describing in 968 has the mutant that reduces another 34rFW1.4 of immunogenic extra displacement in its body.Particularly, the Trimutant that is called 34rFW1.4_VLK50R_DHP comprises Serine (S) on 12 (the AHo numberings) of heavy chain position, comprising Threonine (T) on 103 (the AHo numberings) of heavy chain position and comprise Threonine (T) on 144 (the AHo numberings) of heavy chain position.Carry out the stability study of parental generation and mutant 34rFW1.4 antibody under following condition accelerating.

With 34rFW1.4 antibody, mutant 34rFW1.4_VL_K50R and 34rFW1.4_VLK50R_DHP at phosphate buffered saline(PBS) (50mM Na2HPO4,150mM NaCl, pH6.5) be concentrated in preparation up to 20,40 and 60mg/mL and 40 ℃ of 2 weeks of incubation.34rFW1.4 and 34rFW1.4_K47R antibody are concentrated in phosphate buffered saline(PBS) (50mM Na2HPO4,150mM NaCl, pH6.5) preparation up to 20 and 60mg/mL, and 40 ℃ of lower 2 weeks of incubation.Before the incubation of 14 days and afterwards, under reduction and non-reduced condition, the degraded of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analytic sample of use 12.5%.Before incubation period and afterwards, use size exclusion high performance liquid chromatography (SE-HPLC) to come the monomer content of working sample and solubility to assemble thing.Monomer and non-monomeric species are distinguished TSKgel Super SW2000 post (TOSOH Bioscience) is upper, and be that the area at monomer peak is divided by the total area at all products peak with the percentage calculation of monomeric protein.34rFW1.4 and the result of study of 34rFW1.4_VLK50R_DHP is shown in Fig. 2 A-B, 3A-B and 4A-B.34rFW1.4_VL_K50R the results are shown in Fig. 5 B, 6B and 7B.These test demonstration, and compared to 34rFW1.4,34rFW1.4_VLK50R_DHP has the gathering tendency of minimizing.Mutant 34rFW1.4_K47R also shows such minimizing, as showing in Fig. 8 B and 9B.

In addition, the thermostability and the binding affinity that have compared 34rFW1.4 and 34rFW1.4_VLK50R_DHP antibody.Result shows, compares with parental generation 34rFW1.4 antibody, does not affect stability or in conjunction with active for generation of the sudden change of 34rFW1.4_VLK50R_DHP antibody.

Equivalent

According to above stated specification, the embodiment of many changes of the present invention and change it will be apparent to those skilled in the art that.Therefore, this specification sheets should be construed as merely illustrative, and is used for the best enforcement model of the present invention of instruction those skilled in the art.The details of structure can change, and is not deviating from spirit of the present invention, and keeps to fall the exclusivity use of all changes within the scope of the claims.This invention is intended to only limit to claims and the applicable desired degree of legal provisions.

All documents and the analogous material quoted in the application comprise that integral body is incorporated this paper into by reference clearly for patent, patent application, paper, books, Diplomarbeit, lecture, webpage, accompanying drawing and/or appendix (no matter which kind of form this type of document and analogous material are).If one or more different from the application in the document of incorporating into and analogous material or contradict comprise term, the term usage of definition, technology of description etc., be as the criterion with the application.

Chapter title used herein is only for organizational goal, and never is interpreted as limiting the theme of description.

Although described the present invention in conjunction with various embodiments and embodiment, this instruction is not intended to be defined in this type of embodiment or embodiment.On the contrary, the present invention includes variously substitute, change and equivalent, as skilled in the art to understand.

Unless clearly indicate, otherwise claim should not be read as and is defined in described order or element.Should be appreciated that the various variations that to carry out form and details, and do not deviate from the scope of claims.Therefore, claimed all interior embodiments of scope and spirit that drop on following claim and equivalent thereof.

Claims

1. the antibody of a specific binding human TNF alpha, it comprises:

A. the variable light chain that comprises the sequence of SEQ ID NO:2 or SEQ ID NO:14; With

B. the variable heavy chain that comprises the sequence of SEQ ID NO:5.

2. the antibody of claim 1, wherein said variable light chain comprises the sequence of SEQ ID NO:2.

3. the antibody of claim 1, wherein said variable light chain comprises the sequence of SEQ ID NO:14.

4. the antibody of claim 1, it also has the joint of the sequence that comprises SEQ ID NO:7.

5. the antibody of claim 1, wherein said antibody comprises the sequence of SEQ ID NO:10.

6. the antibody of claim 1, wherein said antibody comprises the sequence of SEQ ID NO:17.

7. pharmaceutical composition, it comprises the antibody of the claim 1 for the treatment of significant quantity, and pharmaceutically acceptable carrier.

8. a method for the treatment of the alpha mediated disease of TNF, comprise the pharmaceutical composition of using claim 7 to the experimenter that these needs are arranged.

9. the method for claim 8, the alpha mediated disease of wherein said TNF are the illnesss of eye, its be selected from uveitis, Behcet's disease, the retinitis, dry eyes, glaucoma,

Syndrome, diabetic neuropathy, scleritis, age related macular degeneration and keratitis.

10. the method for claim 8 is wherein by in eye, nose, through ear, hypogloeeis, use described pharmaceutical composition through skin, part, oral, intranasal, rectum or parenteral.

11. the method for claim 10 is wherein used described pharmaceutical composition to comprise single part of 0.1 to 100mg antibody or broken dose.

12. the method for claim 8, the alpha mediated disease of wherein said TNF is uveitis, and to described experimenter's eye topical application described pharmaceutical composition.

13. the nucleic acid molecule of the separation of the antibody of the claim 1 of encoding.

14. carrier that comprises the nucleic acid molecule of claim 13.

15. host cell that comprises the carrier of claim 14.

16. the antibody of claim 1, wherein said antibody are Fab, Fab', F (ab) ' 2, scFv (scFv), Fv fragment or linear antibody.

17. bivalent or bispecific molecule that comprises the antibody of claim 1.

18. method that is used for the gathering tendency of minimizing antibody, described method comprises introduces the variable light chain (VL) of antibody and the interface of variable heavy chain (VH) with one or more amino-acid substitutions, wherein said one or more displacement is positioned on such residue position, described residue position is selected for the free energy that makes between VL and VH and is reduced by at least 0.5kcal/mol, thereby compared to parental generation antibody, reduce the gathering tendency of antibody.

19. the method for claim 18, wherein said antibody are Fab, Fab', F (ab) ' 2, scFv (scFv), Fv fragment, bispecific antibody, strand bispecific antibody, series connection antibody or linear antibody.

20. the method for claim 18, the sequence of the sequence of the variable light chain of wherein said antibody and SEQ ID NO:1 has at least 65% identity.

21. the method for claim 20, wherein said modification are included in the AHo position 50 of variable light chain and/or the displacement on AHo position 47.

22. the method for claim 21, the arginine (R) on the arginine (R) on the wherein said AHo position that is replaced into variable light chain 50 and/or AHo position 47.

23. the method for claim 22, wherein the Methionin (K) on the AHo position 47 and/or 50 of variable light chain is replaced by arginine (R).

24. the method for claim 18, wherein said antibody have the variable heavy chain sequence that sequence with SEQ ID NO:1 has at least 85% identity.

25. the method for claim 18, wherein said antibody have the variable heavy chain sequence that sequence with the sequence of SEQ ID NO:3 or SEQ ID NO:4 has at least 85% identity.

26. the method for claim 18, it also is included in the amino-acid substitution on the AHo position 12,103 and 144 of variable heavy chain.

27. antibody that produces by the method for claim 18.